With high sensitivity in detecting acute brain events such as seizures, FDG PET can be used as an important tool for neurocutaneous melanosis disease monitoring.

Early recognition of longus colli calcific tendinitis can prevent unnecessary interventions including antibiotics and surgical procedures.

OBJECTIVES:

Standing orders are an effective way to increase vaccination rates, yet little is known about how pediatricians use this strategy for childhood immunizations. We assessed current use of, barriers to using, and factors associated with use of standing orders for vaccination among pediatricians.

Key Points

Localized skin lesions are characteristic of cutaneous leishmaniasis (CL); however, Leishmania (Viannia) species, which are responsible for most CL cases in the Americas, can spread systemically, sometimes resulting in mucosal disease. Detection of Leishmania has been documented in healthy mucosal tissues (conjunctiva, tonsils, and nasal mucosa) and healthy skin of CL patients and in individuals with asymptomatic infection in areas of endemicity of L. (V.) panamensis and L. (V.) braziliensis transmission. However, the conditions and mechanisms that favor parasite persistence in healthy mucosal tissues are unknown. In this descriptive study, we compared the cell populations of the nasal mucosa (NM) of healthy donors and patients with active CL and explored the immune gene expression signatures related to molecular detection of Leishmania in this tissue in the absence of clinical signs or symptoms of mucosal disease. The cellular composition and gene expression profiles of NM samples from active CL patients were similar to those of healthy volunteers, with a predominance of epithelial over immune cells, and within the CD45⁺ cell population, a higher frequency of CD66b⁺ followed by CD14⁺ and CD3⁺ cells. In CL patients with molecular evidence of Leishmania persistence in the NM, genes characteristic of an anti-inflammatory and tissue repair responses (IL4R, IL5RA, POSTN, and SATB1) were overexpressed relative to NM samples from CL patients in which Leishmania was not detected. Here, we report the first immunological description of subclinically infected NM tissues of CL patients and provide evidence of a local anti-inflammatory environment favoring parasite persistence in the NM.

Glaesserella (Haemophilus) parasuis is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer’s disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in G. parasuis virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069cap) through allelic exchange following natural transformation. HS069cap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069cap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069cap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to G. parasuis virulence as well as nasal colonization in pigs.

Staphylococcus aureus is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in S. aureus, and mutations in purR result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing purR mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for de novo purine biosynthesis in this phenotype and indicating that S. aureus in the mammalian host experiences purine limitation. Using cell culture, we showed that while purR mutants are not altered in epithelial cell binding, compared to that of wild-type (WT) S. aureus, purR mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with purR mutants having increased transcription of fnb genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of S. aureus is affected by sensing of purine levels during infection of the mammalian host.

For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9^H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the A56 virus, the G9^H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9^H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9^H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9^H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9^H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.

IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter’s G9 subcomponent. Although the G9^H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.

Governments around the world must rapidly mobilize and make difficult policy decisions to mitigate the coronavirus disease 2019 (COVID-19) pandemic. Because deaths have been concentrated at older ages, we highlight the important role of demography, particularly, how the age structure of a population may help explain differences in fatality rates...

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in...

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N2) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N2 fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we...

Purpose:

To identify specific actions and characteristics of health care providers (HCPs) in the United States and Canada that influenced patients with type 2 diabetes who were initially reluctant to begin insulin.

Growing evidence indicates that oxidative and endoplasmic reticular stress, which trigger changes in ion channels and inflammatory pathways that may undermine cellular homeostasis and survival, are critical determinants of injury in the diabetic kidney. Cells are normally able to mitigate these cellular stresses by maintaining high levels of autophagy, an intracellular lysosome-dependent degradative pathway that clears the cytoplasm of dysfunctional organelles. However, the capacity for autophagy in both podocytes and renal tubular cells is markedly impaired in type 2 diabetes, and this deficiency contributes importantly to the intensity of renal injury. The primary drivers of autophagy in states of nutrient and oxygen deprivation—sirtuin-1 (SIRT1), AMP-activated protein kinase (AMPK), and hypoxia-inducible factors (HIF-1α and HIF-2α)—can exert renoprotective effects by promoting autophagic flux and by exerting direct effects on sodium transport and inflammasome activation. Type 2 diabetes is characterized by marked suppression of SIRT1 and AMPK, leading to a diminution in autophagic flux in glomerular podocytes and renal tubules and markedly increasing their susceptibility to renal injury. Importantly, because insulin acts to depress autophagic flux, these derangements in nutrient deprivation signaling are not ameliorated by antihyperglycemic drugs that enhance insulin secretion or signaling. Metformin is an established AMPK agonist that can promote autophagy, but its effects on the course of CKD have been demonstrated only in the experimental setting. In contrast, the effects of sodium-glucose cotransporter–2 (SGLT2) inhibitors may be related primarily to enhanced SIRT1 and HIF-2α signaling; this can explain the effects of SGLT2 inhibitors to promote ketonemia and erythrocytosis and potentially underlies their actions to increase autophagy and mute inflammation in the diabetic kidney. These distinctions may contribute importantly to the consistent benefit of SGLT2 inhibitors to slow the deterioration in glomerular function and reduce the risk of ESKD in large-scale randomized clinical trials of patients with type 2 diabetes.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K_1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue–absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical “second” cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

Multidrug resistance (MDR) in cancer arises from cross-resistance to structurally- and functionally-divergent chemotherapeutic drugs. In particular, MDR is characterized by increased expression and activity of ATP-binding cassette (ABC) superfamily transporters. Sphingolipids are substrates of ABC proteins in cell signaling, membrane biosynthesis, and inflammation, for example, and their products can favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key regulatory enzyme encoded by the UDP-glucose ceramide glucosyltransferase (UGCG) gene. Stressed cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after UGCG mediates incorporation into GlcCer. Given that cancer cells seem to mobilize UGCG and have increased GSL content for ceramide clearance, which ultimately contributes to chemotherapy failure, here we investigated how inhibition of GSL biosynthesis affects the MDR phenotype of chronic myeloid leukemias. We found that MDR is associated with higher UGCG expression and with a complex GSL profile. UGCG inhibition with the ceramide analog d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4) greatly reduced GSL and monosialotetrahexosylganglioside levels, and co-treatment with standard chemotherapeutics sensitized cells to mitochondrial membrane potential loss and apoptosis. ABC subfamily B member 1 (ABCB1) expression was reduced, and ABCC-mediated efflux activity was modulated by competition with nonglycosylated ceramides. Consistently, inhibition of ABCC-mediated transport reduced the efflux of exogenous C6-ceramide. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, which typically overcomes drug resistance through distinct mechanisms. This work sheds light on the involvement of GSL in chemotherapy failure, and its findings suggest that targeted GSL modulation could help manage MDR leukemias.

Triple-negative breast cancer (TNBC) is characterized by its aggressive biology, early metastatic spread, and poor survival outcomes. TNBC lacks expression of the targetable receptors found in other breast cancer subtypes, mandating use of cytotoxic chemotherapy. However, resistance to chemotherapy is a significant problem, encountered in about two-thirds of TNBC patients, and new strategies are needed to mitigate resistance. In this issue of the Journal of Biological Chemistry, Geck et al. report that TNBC cells are highly sensitive to inhibition of the de novo polyamine synthesis pathway and that inhibition of this pathway sensitizes cells to TNBC-relevant chemotherapy, uncovering new opportunities for addressing chemoresistance.

Premature infants have a higher incidence of indirect hyperbilirubinemia than term infants. Management of neonatal indirect hyperbilirubinemia in late preterm and term neonates has been well addressed by recognized, consensus-based guidelines. However, the extension of these guidelines to the preterm population has been an area of uncertainty because of limited evidence. This leads to variation in clinical practice and lack of recognition of the spectrum of bilirubin-induced neurologic dysfunction (BIND) in this population. Preterm infants are metabolically immature and at higher risk for BIND at lower bilirubin levels than their term counterparts. Early use of phototherapy to eliminate BIND and minimize the need for exchange transfusion is the goal of treatment in premature neonates. Although considered relatively safe, phototherapy does have side effects, and some NICUs tend to overuse phototherapy. In this review, we describe the epidemiology and pathophysiology of BIND in preterm neonates, and discuss our approach to standardized management of indirect hyperbilirubinemia in the vulnerable preterm population. The proposed treatment charts suggest early use of phototherapy in preterm neonates with the aim of reducing exposure to high irradiance levels, minimizing the need for exchange transfusions, and preventing BIND. The charts are pragmatic and have additional curves for stopping phototherapy and escalating its intensity. Having a standardized approach would support future research and quality improvement initiatives that examine dose and duration of phototherapy exposure with relation to outcomes.

Mechanical heterogeneity of a sedimentary sequence exerts a primary control on the geometry of fault zones and the proportion of offset accommodated by folding. The Wildensbuch Fault Zone in the Swiss Molasse Basin, with a maximum throw of 40 m, intersects a Mesozoic section containing a thick (120 m) clay-dominated unit (Opalinus Clay) over- and underlain by more competent limestone units. Interpretation of a 3D seismic reflection survey indicates that the fault zone formed by upward propagation of an east–west-trending basement structure, through the Mesozoic section, in response to NE–SW Miocene extension. This configuration formed an array of left-stepping normal fault segments above and below the Opalinus Clay. In cross-section a broad monoclinal fold is observed in the Opalinus Clay. Folding, however, is not ubiquitous and occurs in the Opalinus Clay where fault segments above and below are oblique to one another; where they are parallel the fault passes through the Opalinus Clay with little folding. These observations demonstrate that, even in strongly heterogeneous sequences, here a four-fold difference in both Young's modulus and cohesion between layers, the occurrence of folding may depend on the local relationship between fault geometry and applied stress field rather than rheological properties alone.

Reactive immunization with a single-dose cholera vaccine that could rapidly (within days) protect immunologically naive individuals during virgin soil epidemics, when cholera reaches immunologically naive populations that have not experienced cholera for decades, would facilitate cholera control. One dose of attenuated Vibrio cholerae O1 classical Inaba vaccine CVD 103-HgR (Vaxchora) containing ≥2 x 10⁸ CFU induces vibriocidal antibody seroconversion (a correlate of protection) in >90% of U.S. adults. A previous CVD 103-HgR commercial formulation required ≥2 x 10⁹ CFU to elicit high levels of seroconversion in populations in developing countries. We compared the vibriocidal responses of Malians (individuals 18 to 45 years old) randomized to ingest a single ≥2 x 10⁸-CFU standard dose (n = 50) or a ≥2 x 10⁹-CFU high dose (n = 50) of PaxVax CVD 103-HgR with buffer or two doses (n = 50) of Shanchol inactivated cholera vaccine (the immunologic comparator). To maintain blinding, participants were dosed twice 2 weeks apart; CVD 103-HgR recipients ingested placebo 2 weeks before or after ingesting vaccine. Seroconversion (a ≥4-fold vibriocidal titer rise) between the baseline and 14 days after CVD 103-HgR ingestion and following the first and second doses of Shanchol were the main outcomes measured. By day 14 postvaccination, the rates of seroconversion after ingestion of a single standard dose and a high dose of CVD 103-HgR were 71.7% (33/46 participants) and 83.3% (40/48 participants), respectively. The rate of seroconversion following the first dose of Shanchol, 56.0% (28/50 participants), was significantly lower than that following the high dose of CVD 103-HgR (P = 0.003). The vibriocidal geometric mean titer (GMT) of the high dose of CVD 103-HgR exceeded the GMT of the standard dose at day 14 (214 versus 95, P = 0.045) and was ~2-fold higher than the GMT on day 7 and day 14 following the first Shanchol dose (P > 0.05). High-dose CVD 103-HgR is recommended for accelerated evaluation in developing countries to assess its efficacy and practicality in field situations. (This study has been registered at ClinicalTrials.gov under registration no. NCT02145377.)

In 2015, the M_w 7.8 Gorkha earthquake struck Nepal, triggering thousands of landslides across the central and eastern Himalayas. These landslides had many adverse effects, including causing widespread damage to low-grade transport routes (e.g. tracks, footpaths) in rural regions that depend on tourism for survival. Langtang Valley is a glacial–periglacial landscape located 60 km north of Kathmandu. It is one of the most popular trekking regions in Nepal and has been severely affected by Gorkha earthquake-triggered and monsoon-triggered landsliding. Here, qualitative and quantitative observations from fieldwork and remote sensing are used to describe the materials and geomorphology of the landslides across Langtang Valley, and to quantify the extent to which coseismic and monsoon-triggered landslides have affected Langtang's trekking infrastructure. The dominant bedrock materials involved within Langtang landslides are found to be a range of gneisses and intruded leucogranites. In total, 64 landslides are found to have intersected trekking paths across Langtang, with coseismic and monsoon-triggered landslides having an impact on c. 3 km and 0.8 km of path respectively. It is observed that the practice of reconstructing paths through unstable landslide deposits is leaving the trekking infrastructure across Langtang increasingly vulnerable to future failure.

Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

Human herpesvirus 6 (HHV-6) is an important cause of meningitis and meningoencephalitis. As testing for HHV-6 in cerebrospinal fluid (CSF) is more readily available using the FilmArray Meningitis/Encephalitis panel (FA-ME; BioFire Diagnostics, Salt Lake City, UT), we aimed to determine the clinical significance of detecting HHV-6 in order to identify true infections and to ensure appropriate antiviral initiation. Chart review on 25 patients positive for HHV-6 by FA-ME was performed to determine clinical presentation, comorbidity, treatment, and outcome. The presence of chromosomally integrated HHV-6 (ciHHV-6) DNA was also investigated. Of 1,005 children tested by FA-ME, HHV-6 was detected in 25 (2.5%). Five patients were diagnosed with either HHV-6 meningitis or meningoencephalitis based on HHV-6 detection in CSF, clinical presentation, and radiographic findings. Detection of HHV-6 by FA-ME led to discontinuation of acyclovir within 12.0 h in all 12 patients empirically treated with acyclovir. Six of the 12 patients were started on ganciclovir therapy within 6.8 h; 4 of these were treated specifically for HHV-6 infection, whereas therapy was discontinued in the remaining 2 patients. CSF parameters were not generally predictive of HHV-6 positivity. The presence of ciHHV-6 was confirmed in 3 of 18 patients who could be tested. Five of the 25 patients included in the study were diagnosed with HHV-6 meningitis/meningoencephalitis. FA-ME results led to discontinuation of empirical antiviral treatment in 12 patients and appropriate initiation of ganciclovir in 4 patients. In our institution, detection of HHV-6 using FA-ME led to faster establishment of disease etiology and optimization of antimicrobial therapy.

Retrospective lineage tracing harnesses naturally occurring mutations in cells to elucidate single cell development. Common single-cell phylogenetic fate mapping methods have utilized highly mutable microsatellite loci found within the human genome. Such methods were limited by the introduction of in vitro noise through polymerase slippage inherent in DNA amplification, which we characterized to be approximately 10–100x higher than the in vivo replication mutation rate. Here, we present RETrace, a method for simultaneously capturing both microsatellites and methylation-informative cytosines to characterize both lineage and cell type, respectively, from the same single cell. An important unique feature of RETrace was the introduction of linear amplification of microsatellites in order to reduce in vitro amplification noise. We further coupled microsatellite capture with single-cell reduced representation bisulfite sequencing (scRRBS), to measure the CpG methylation status on the same cell for cell type inference. When compared to existing retrospective lineage tracing methods, RETrace achieved higher accuracy (88% triplet accuracy from an ex vivo HCT116 tree) at a higher cell division resolution (lowering the required number of cell division difference between single cells by approximately 100 divisions). Simultaneously, RETrace demonstrated the ability to capture on average 150,000 unique CpGs per single cell in order to accurately determine cell type. We further formulated additional developments that would allow high-resolution mapping on microsatellite-stable cells or tissues with RETrace. Overall, we present RETrace as a foundation for multi-omics lineage mapping and cell typing of single cells.

The microtubule-binding taxanes, docetaxel and cabazitaxel, are administered intravenously for the treatment of castration-resistant prostate cancer (CRPC) as the oral administration of these drugs is largely hampered by their low and highly variable bioavailabilities. Using a simple, rapid, and environmentally friendly microwave-assisted protocol, we have synthesized a number of 3,5-bis(styryl)pyrazoles 2a-l, thus allowing for their screening for antiproliferative activity in the androgen-independent PC3 prostate cancer cell line. Surprisingly, two of these structurally simple 3,5-bis(styryl)pyrazoles (2a and 2l) had concentrations which gave 50% of the maximal inhibition of cell proliferation (GI₅₀) in the low micromolar range in the PC3 cell line and were thus selected for extensive further biologic evaluation (apoptosis and cell cycle analysis, and effects on tubulin and microtubules). Our findings from these studies show that 3,5-bis[(1E)-2(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l 1) caused significant effects on the cell cycle in PC3 cells, with the vast majority of treated cells in the G₂/M phase (89%); 2) induces cell death in PC3 cells even after the removal of the compound; 3) binds to tubulin [dissociation constant (K_d) 0.4 ± 0.1 μM] and inhibits tubulin polymerization in vitro; 4) had no effect upon the polymerization of the bacterial cell division protein FtsZ (a homolog of tubulin); 5) is competitive with paclitaxel for binding to tubulin but not with vinblastine, crocin, or colchicine; and 6) leads to microtubule depolymerization in PC3 cells. Taken together, these results suggest that 3,5-bis(styryl)pyrazoles warrant further investigation as lead compounds for the treatment of CRPC.

The 78-kDa glucose-regulated protein (GRP78), an endoplasmic reticulum (ER) chaperone, is a master regulator of the ER stress. A number of studies revealed that high levels of GRP78 protein in cancer cells confer multidrug resistance (MDR) to therapeutic treatment. Therefore, drug candidate that reduces GRP78 may represent a novel approach to eliminate MDR cancer cells. Our earlier studies showed that a set of 4H-chromene derivatives induced selective cytotoxicity in MDR cancer cells. In the present study, we elucidated its selective mechanism in four MDR cancer cell lines with one lead candidate (CXL146). Cytotoxicity results confirmed the selective cytotoxicity of CXL146 toward the MDR cancer cell lines. We noted significant overexpression of GRP78 in all four MDR cell lines compared with the parental cell lines. Unexpectedly, CXL146 treatment rapidly and dose-dependently reduced GRP78 protein in MDR cancer cell lines. Using human leukemia (HL) 60/mitoxantrone (MX) 2 cell line as the model, we demonstrated that CXL146 treatment activated the unfolded protein response (UPR); as evidenced by the activation of inositol-requiring enzyme 1α, protein kinase R–like ER kinase, and activating transcription factor 6. CXL146-induced UPR activation led to a series of downstream events, including extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase activation, which contributed to CXL146-induced apoptosis. Targeted reduction in GRP78 resulted in reduced sensitivity of HL60/MX2 toward CXL146. Long-term sublethal CXL146 exposure also led to reduction in GRP78 in HL60/MX2. These data collectively support GRP78 as the target of CXL146 in MDR treatment. Interestingly, HL60/MX2 upon long-term sublethal CXL146 exposure regained sensitivity to mitoxantrone treatment. Therefore, further exploration of CXL146 as a novel therapy in treating MDR cancer cells is warranted.

The aim of this study was to evaluate the efficacy of ¹⁷⁷Lu-prostate-specific membrane antigen (PSMA)-617 (¹⁷⁷Lu-PSMA) and selective internal radiation therapy (SIRT) for the treatment of liver metastases of castration-resistant prostate cancer. Methods: Safety and survival of patients with metastatic castration-resistant prostate cancer and liver metastases assigned to ¹⁷⁷Lu-PSMA alone (n = 31) or in combination with SIRT (n = 5) were retrospectively analyzed. Additionally, a subgroup (n = 10) was analyzed using morphologic and molecular response criteria. Results: Median estimated survival was 5.7 mo for ¹⁷⁷Lu-PSMA alone and 8.4 mo for combined sequential ¹⁷⁷Lu-PSMA and SIRT. ¹⁷⁷Lu-PSMA achieved discordant therapy responses with both regressive and progressive liver metastases in the same patient (best vs. worst responding metastases per patient: –35% vs. +63% diameter change; P < 0.05). SIRT was superior to ¹⁷⁷Lu-PSMA for the treatment of liver metastases (0% vs. 56% progression). Conclusion: The combination of ¹⁷⁷Lu-PSMA and SIRT is efficient and feasible for the treatment of advanced prostate cancer. ¹⁷⁷Lu-PSMA alone seems to have limited response rates in the treatment of liver metastases.

Prostate-specific membrane antigen (PSMA)–targeting α-radiation therapy (TAT) is an emerging treatment modality for metastatic castration-resistant prostate cancer. There is a subgroup of patients with poor response despite sufficient expression of PSMA in their tumors. The aim of this work was to characterize PSMA-TAT–nonresponding lesions by targeted next-generation sequencing. Methods: Of 60 patients treated with ²²⁵Ac-PSMA-617, we identified 10 patients who presented with a poor response despite sufficient tumor uptake in PSMA PET/CT. We were able to perform CT-guided biopsies with histologic validation of the nonresponding lesions in 7 of these nonresponding patients. Specimens were analyzed by targeted next-generation sequencing interrogating 37 DNA damage-repair–associated genes. Results: In the 7 tumor samples analyzed, we found a total of 15 whole-gene deletions, deleterious or presumably deleterious mutations affecting TP53 (n = 3), CHEK2 (n = 2), ATM (n = 2), and BRCA1, BRCA2, PALB2, MSH2, MSH6, NBN, FANCB, and PMS1 (n = 1 each). The average number of deleterious or presumably deleterious mutations was 2.2 (range, 0–6) per patient. In addition, several variants of unknown significance in ATM, BRCA1, MSH2, SLX4, ERCC, and various FANC genes were detected. Conclusion: Patients with resistance to PSMA-TAT despite PSMA positivity frequently harbor mutations in DNA damage-repair and checkpoint genes. Although the causal role of these alterations in the patient outcome remains to be determined, our findings encourage future studies combining PSMA-TAT and DNA damage-repair–targeting agents such as poly(ADP-ribose)-polymerase inhibitors.

For effective clinical management of patients being treated with ²²³Ra, there is a need for radiographic response biomarkers to minimize disease progression and to stratify patients for subsequent treatment options. The objective of this study was to evaluate an automated bone scan index (aBSI) as a quantitative assessment of bone scans for radiographic response in patients with metastatic castration-resistant prostate cancer (mCRPC). Methods: In a multicenter retrospective study, bone scans from patients with mCRPC treated with monthly injections of ²²³Ra were collected from 7 hospitals in Sweden. Patients with available bone scans before treatment with ²²³Ra and at treatment discontinuation were eligible for the study. The aBSI was generated at baseline and at treatment discontinuation. The Spearman rank correlation was used to correlate aBSI with the baseline covariates: alkaline phosphatase (ALP) and prostate-specific antigen (PSA). The Cox proportional-hazards model and Kaplan–Meier curve were used to evaluate the association of covariates at baseline and their change at treatment discontinuation with overall survival (OS). The concordance index (C-index) was used to evaluate the discriminating strength of covariates in predicting OS. Results: Bone scan images at baseline were available from 156 patients, and 67 patients had both a baseline and a treatment discontinuation bone scan (median, 5 doses; interquartile range, 3–6 doses). Baseline aBSI (median, 4.5; interquartile range, 2.4–6.5) was moderately correlated with ALP (r = 0.60, P < 0.0001) and with PSA (r = 0.38, P = 0.003). Among baseline covariates, aBSI (P = 0.01) and ALP (P = 0.001) were significantly associated with OS, whereas PSA values were not (P = 0.059). After treatment discontinuation, 36% (24/67), 80% (54/67), and 13% (9/67) of patients demonstrated a decline in aBSI, ALP, and PSA, respectively. As a continuous variable, the relative change in aBSI after treatment, compared with baseline, was significantly associated with OS (P < 0.0001), with a C-index of 0.67. Median OS in patients with both aBSI and ALP decline (median, 134 wk) was significantly longer than in patients with ALP decline only (median, 77 wk; P = 0.029). Conclusion: Both aBSI at baseline and its change at treatment discontinuation were significant parameters associated with OS. The study warrants prospective validation of aBSI as a quantitative imaging response biomarker to predict OS in patients with mCRPC treated with ²²³Ra.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAF^V600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRAS^Q61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAF^V600E PDX highlighting its effectiveness in combating the advent of drug resistance.

Editor’s Note: This article is adapted from the address Ms. Valentine delivered as the recipient of the American Diabetes Association’s (ADA’s) Outstanding Educator in Diabetes Award for 2019. She delivered the address in June 2019 at the Association’s 79th Scientific Sessions in San Francisco, CA. A webcast of this speech is available for viewing at the ADA website (professional.diabetes.org/webcast/outstanding-educator-diabetes-award-lecture%E2%80%94-most-important-thing-we-give-people-hope).

Despite significant advances in therapies for pediatric type 1 diabetes, achievement of glycemic targets remains elusive, and management remains burdensome for patients and their families. This article identifies common challenges in diabetes management at the patient-provider and health care system levels and proposes practical approaches to overcoming therapeutic inertia to enhance health outcomes for youth with type 1 diabetes.

ABSTRACT

The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (⁷⁶⁷DKE⁷⁶⁹) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K⁷⁶⁸ was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation.

IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K⁷⁶⁸ in LC translocation. K⁷⁶⁸ was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K⁷⁶⁸ did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K⁷⁶⁸ is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.

Among the emerging techniques to detect the real footprint of buried ore deposits is isotope tracing. Novel and automated preparation systems such as continuous flow isotope ratio mass spectrometry, off-axis integrated cavity output spectroscopy for isotopic compositions of selected molecules, multi-collector inductively coupled-plasma mass spectrometry (ICP-MS), triple quadrupole ICP-MS, laser ablation ICP-MS, and a multitude of inline preparation systems have facilitated the use of isotopes as tracers in mineral exploration, as costs for isotope analyses have decreased and the time required for the analyses has improved. In addition, the isotope systems being used have expanded beyond the traditional light stable and Pb isotopes to include a multitude of elements that behave differently during processes that promote the mobilization of elements during both primary and secondary dispersion. Isotopes are also being used to understand barren areas that lack a critical process to form an ore deposit and to reveal precise redox mechanisms. The goal is to be able to use isotopes to reflect a definitive process that occurs in association with the deposit and not in barren systems, and then to relate these to something that is easier to measure, namely elemental concentrations. As new generations of exploration and environmental scientists are becoming more comfortable with the application of isotopes to effectively trace processes involved in geoscience, and new technologies for rapid and inexpensive analyses of isotopes are continually being developed, novel applications of isotope tracing are becoming more mainstream.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

For manufacturers of both sterile and nonsterile pharmaceuticals, there is an expectation that the manufacturing process is performed in a manner that prevents extraneous contamination so that the products are provided in a safe, integral, pure, and unadulterated form. As part of that process, cleaning and disinfection are an absolute necessity. Although cleaning and disinfection support control of microbial contamination through preventive and corrective action, specific compendia methods do not currently exist. The intent of this paper is to provide a general guidance on how to perform disinfectant efficacy validation and implementation. This includes how to make sure the concepts are understood, how to interpret facility data and utilize it to demonstrate control awareness for your facilities, and how to leverage the data to reduce redundancies in validation or verification. This paper represents the thoughts and best practices of the authoring team and their respective companies and provides an efficient way to qualify disinfectants without impacting the quality of the study. If you choose to follow the recommendations in this paper, you must ensure that the appropriate rationale is sound and the scientific data is documented. It is the belief of the authoring team that only then will this approach meet regulatory requirements.

ABSTRACTThe Standard Days Method (SDM), a modern fertility awareness-based family planning method, has been introduced in 30 countries since its development in 2001. It is still unclear to what extent the SDM was mainstreamed within the family planning method mix, particularly in low- and middle-income country (LMIC) settings, where the SDM had been introduced by donors and implementing partners. This review of implementation science publications on the SDM in LMICs first looked at community pilot studies of the SDM to determine the acceptability of the method; correct use and efficacy rates; demographics of users; and changes to contraceptive prevalence rates and family planning behaviors, especially among men and couples. Then, we examined the status of the SDM in the 16 countries that had attempted to scale up the method within national family planning protocols, training, and service delivery. At the community level, evidence demonstrated a high level of acceptability of the method; efficacy rates comparable to the initial clinical trials; diversity in demographic characteristics of users, including first-time or recently discontinued users of family planning; increased male engagement in family planning; and improved couple's communication. Nationally, few countries had scaled up the SDM due to uneven stakeholder engagement, lackluster political will, and competing resource priorities. Results of this review could help policy makers determine the added value of the SDM in the contraceptive method mix and identify potential barriers to its implementation moving forward.

ABSTRACTBackground:A significant number of girls are married as children, which negatively impacts their health, education, and development. Given the sheer numbers of girls at risk of child marriage globally, the challenge to eliminate the practice is daunting. Programs to prevent child marriage are typically small-scale and overlook the costs and scalability of the intervention.Implementation:This study tested and costed different approaches to preventing child marriage in rural Burkina Faso and Tanzania. The approaches tested were community dialogue, provision of school supplies, provision of a livestock asset, a model including all components, and a control arm. A quasi-experimental design was employed with surveys undertaken at baseline and after 2 years of intervention. We examined the prevalence of child marriage and school attendance controlling for background characteristics and stratified by age group. Programmatic costs were collected prospectively.Results:Among those in the community dialogue arm in Burkina Faso, girls aged 15 to 17 years had two-thirds less risk (risk ratio [RR]=0.33; 95% confidence interval [CI]=0.19, 0.60) of being married and girls aged 12 to 14 years had a greater chance of being in school (RR=1.18; 95% CI=1.07,1.29) compared to the control site. In Tanzania, girls aged 12 to 14 years residing in the multicomponent arm had two-thirds less risk of being married (RR=0.33; 95% CI=0.11, 0.99), and girls 15 to 17 in the conditional asset location had half the risk (RR=0.52; 95% CI=0.30, 0.91). All the interventions tested in Tanzania were associated with increased risk of girls 12 to 14 years old being in school, and the educational promotion arm was also associated with a 30% increased risk of girls aged 15 to 17 years attending school (RR=1.3; 95% CI=1.01, 1.67). Costs per beneficiary ranged from US$9 to US$117.Conclusion:The study demonstrates that minimal, low-cost approaches can be effective in delaying child marriage and increasing school attendance. However, community dialogues need to be designed to ensure sufficient quality and intensity of messaging. Program managers should pay attention to the cost, quality, and coverage of interventions, especially considering that child marriage persists in the most hard-to-reach rural areas of many countries.

Patients with systemic immunoglobulin light chain amyloidosis (AL) with no evidence of cardiac involvement by consensus criteria have excellent survival, but 20% will die within 5 years of diagnosis and prognostic factors remain poorly characterised. We report the outcomes of 378 prospectively followed Mayo stage I patients (N-terminal pro b-type natriuretic peptide <332 ng/L, high sensitivity cardiac troponin <55 ng/L). The median presenting N-terminal pro b-type natriuretic peptide was 161 ng/L, high sensitivity cardiac troponin 10 ng/L, creatinine 76 μmol/L and mean left ventricular septal wall thickness, 10 mm. Median follow up was 42 (1-117 months), with 71 deaths; median overall survival was not reached (78% survival at 5 years). Although no patients had cardiac involvement by echocardiogram, a proportion (n=25/90, 28%) had cardiac involvement by cardiac magnetic resonance imaging. Age, autonomic nervous system involvement, N-terminal pro b-type natriuretic peptide >152 ng/L, high sensitivity cardiac troponin >10 ng/L and cardiac involvement by magnetic resonance imaging were predictive for survival; on multivariate analysis only N-terminal pro b-type natriuretic peptide >152 ng/L (P<0.008, hazard ratio [HR] 3.180, confidence interval [CI]: 1.349-7.495) and cardiac involvement on magnetic resonance imaging (P=0.026, HR=5.360, CI: 1.219-23.574) were prognostic. At 5 years, 70% of patients with N-terminal pro b-type natriuretic peptide >152 ng/L were alive. In conclusion, N-terminal pro b-type natriuretic peptide is prognostic for survival in patients with no cardiac involvement by consensus criteria and cardiac involvement is detected by magnetic resonance imaging in such cases. This suggests that N-terminal pro b-type natriuretic peptide thresholds for cardiac involvement in AL may need to be redefined.

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.

Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia (T-ALL), mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-ALL biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of NOTCH receptors by inhibitory antibodies. We previously demonstrated that the NOTCH1-specific neutralizing antibody OMP52M51 prolongs survival in TALL patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-ALL cells resistant to NOTCH1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired NOTCH1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors.

Progression from the initial vascular response upon hyperglycemia to a proliferative stage with neovacularizations is the hallmark of proliferative diabetic retinopathy. Here, we report on the novel diabetic pdx1^–/– zebrafish mutant as a model for diabetic retinopathy that lacks the transcription factor pdx1 through CRISPR-Cas9–mediated gene knockout leading to disturbed pancreatic development and hyperglycemia. Larval pdx1^–/– mutants prominently show vasodilation of blood vessels through increased vascular thickness in the hyaloid network as direct developmental precursor of the adult retinal vasculature in zebrafish. In adult pdx1^–/– mutants, impaired glucose homeostasis induces increased hyperbranching and hypersprouting with new vessel formation in the retina and aggravation of the vascular alterations from the larval to the adult stage. Both vascular aspects respond to antiangiogenic and antihyperglycemic pharmacological interventions in the larval stage and are accompanied by alterations in the nitric oxide metabolism. Thus, the pdx1^–/– mutant represents a novel model to study mechanisms of hyperglycemia-induced retinopathy wherein extensive proangiogenic alterations in blood vessel morphology and metabolic alterations underlie the vascular phenotype.

Approximately 40% of patients with diabetic macular edema (DME) are resistant to anti–vascular endothelial growth factor (VEGF) therapy (rDME). Here, we demonstrate that significant correlations between inflammatory cytokines and VEGF, as observed in naive DME, are lost in patients with rDME. VEGF overexpression in the mouse retina caused delayed inflammatory cytokine upregulation, monocyte/macrophage infiltration (CD11b⁺ Ly6C⁺ CCR2⁺ cells), macrophage/microglia activation (CD11b⁺ CD80⁺ cells), and blood-retinal barrier disruption due to claudin-5 redistribution, which did not recover with VEGF blockade alone. Phosphorylated protein analysis of VEGF-overexpressed retinas revealed rho-associated coiled-coil–containing protein kinase (ROCK) activation. Administration of ripasudil, a selective ROCK inhibitor, attenuated retinal inflammation and claudin-5 redistribution. Ripasudil also contributed to the stability of claudin-5 expression by both transcriptional enhancement and degradation suppression in inflammatory cytokine–stimulated endothelium. Notably, the anti-VEGF agent and the ROCK inhibitor were synergic in suppressing cytokine upregulation, monocyte/macrophage infiltration, macrophage/microglia activation, and claudin-5 redistribution. Furthermore, in vitro analysis confirmed that claudin-5 redistribution depends on ROCK2 but not on ROCK1. This synergistic effect was also confirmed in human rDME cases. Our results suggest that ROCK-mediated claudin-5 redistribution by inflammation is a key mechanism in the anti-VEGF resistance of DME.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3⁺ Treg-cell expansion in the spleen of wild-type mice but not in Gitr^–/– mice. G3C/cps also induced the expansion of nonconventional Cd4⁺Cd25^–/lowFoxp3^lowGitr^int/high (GITR single-positive [sp]) Tregs. Both Cd4⁺Cd25⁺Gitr^highFoxp3⁺ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene–induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in β-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.

Inadequate insulin secretion in response to glucose is an important factor for β-cell failure in type 2 diabetes (T2D). Although HMG-CoA reductase degradation 1 (HRD1), a subunit of the endoplasmic reticulum–associated degradation complex, plays a pivotal role in β-cell function, HRD1 elevation in a diabetic setting contributes to β-cell dysfunction. We report in this study the excessive HRD1 expression in islets from humans with T2D and T2D mice. Functional studies reveal that β-cell–specific HRD1 overexpression triggers impaired insulin secretion that will ultimately lead to severe hyperglycemia; by contrast, HRD1 knockdown improves glucose control and response in diabetic models. Proteomic analysis results reveal a large HRD1 interactome, which includes v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), a master regulator of genes implicated in the maintenance of β-cell function. Furthermore, mechanistic assay results indicate that HRD1 is a novel E3 ubiquitin ligase that targets MafA for ubiquitination and degradation in diabetic β-cells, resulting in cytoplasmic accumulation of MafA and in the reduction of its biological function in the nucleus. Our results not only reveal the pathological importance of excessive HRD1 in β-cell dysfunction but also establish the therapeutic importance of targeting HRD1 in order to prevent MafA loss and suppress the development of T2D.