The Lower and Middle Ordovician of the Yangtze Platform, China, is characterized by a sedimentary succession dominated by carbonate rocks. Three sections spanning the Nantsinkuan/Lunshan, Fenhsiang, Hunghuayuan, and Dawan/Zitai formations, corresponding to the Tremadocian–Dapingian in age, have been sampled for high-resolution ¹³C chemostratigraphy (542 samples in total). Our new ¹³C data reveal five tie-points with the potential for global correlation: (1) a positive ¹³C excursion in the lower Nantsinkuan Formation within the Tremadocian Rossodus manitouensis Zone; (2) an excursion with two peaks roughly within the late Tremadocian Paltodus ‘deltifer’ Zone; (3) a positive ¹³C shift in the lower Hunghuayuan Formation, within the early Floian Serratognathus diversus Zone; (4) a gradual positive ¹³C shift in the late Floian, ranging from the uppermost S. diversus Zone to the basal Oepikodus evae Zone; (5) a minor negative shift in the lower Dawan/Zitai Formation, within the early Dapingian Baltoniodus triangularis Zone. These excursions are herein used for correlation of the Yangtze Platform strata with successions from South China, North China, the Argentine Precordillera, North America and Baltica. From a palaeogeographical perspective the Gudongkou, Xiangshuidong and Daling sections represent depositional environments along an inner to outer ramp profile. ¹³C data from these sections show successively heavier (higher) ¹³C values with increasing depositional depth. This is interpreted as due to remineralization of organic carbon within the carbonate rocks.

Supplementary material: Carbon and oxygen isotope data are available at: https://doi.org/10.6084/m9.figshare.c.4767080

The Northern Paraguai Belt, at the SE border of the Amazonian Craton, central Brazil, has been interpreted as a Brasiliano–Pan-African (c. 650–600 Ma) belt with a foreland basin, recording collisional polyphase tectonism and greenschist-facies metamorphism extending from the late Precambrian to the Cambrian–Ordovician. New structural investigations indicate that the older metasedimentary rocks of the Cuiabá Group represent a Tonian–Cryogenian basement assemblage deformed in two contemporaneous fault-bounded structural sub-domains of wrench-dominated (rake <10°) and contraction-dominated (rake ~30–40°) sinistral transpression, with tectonic vergence towards the SE. The younger late Cryogenian to early Cambrian sedimentary rocks lying to the NW of the Cuiabá Group are non-metamorphic and display only pervasive brittle transtension characterized by normal-oblique faults, fractures and forced drag folds with no consistent vergence pattern. Our analyses suggest that an unconformity separates the metasedimentary Cuiabá Group basement of the Northern Paraguai Belt from the unmetamorphosed sedimentary cover. It is proposed that the latter units were deposited during a post-glacial marine transgression (after c. 635 Ma) in a post-collisional basin. The Paraguai Belt basement and its post-collisional sedimentary cover share a number of significant geological similarities with sequences in the Bassarides Belt and Taoudéni Basin in the SW portion of the West African Craton.

The Tabaquito batholith (Frontal Cordillera, western Argentina), is mainly composed of shallowly emplaced granodiorite to minor monzogranite with abundant mafic microgranular enclaves. New sensitive high-resolution ion microprobe U–Pb zircon ages of c. 337 Ma (biotite granodiorite) and c. 284 Ma (mafic dyke) along with previously published geochronological data suggest that a long-lived magmatic system formed through at least two magmatic pulses at c. 337 and c. 322 Ma with later superimposition of Permian magmatism. The Tabaquito granitoids are metaluminous, calc-alkalic and magnesian with I-type affinity. Elevated Th/Nb, Y/Nb and La/Nb ratios along with negative Nb–Ta and positive Pb anomalies are consistent with a continental arc setting. Hf, Nd and Sr isotopic composition of the Tabaquito granitoids suggests that their source could result from mixing of an old felsic crustal component and a juvenile mafic to intermediate component. New geochronological and geochemical data together with published data reveal a continuous arc setting from the Carboniferous to the Permian in Argentina, and important magmatic compositional variations through time and space controlled by episodic fluctuations in the subduction angle of the oceanic plate. Reported and compiled data allow us to infer the continuity of the Carboniferous magmatic arc along the west margin of Gondwana.

Supplementary material: Detailed petrography, analytical methods and data, zircon cathodoluminescence images and supplementary figures are available at https://doi.org/10.6084/m9.figshare.c.4763993

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.)

Anaplastic lymphoma kinase (ALK) inhibitors have been used in patients with non-small cell lung cancer (NSCLC) harboring EML4-ALK fusion gene.¹ Severe skeletal muscle adverse events of ALK inhibitors, such as muscle weakness, have seldom been reported.^2,3 Herein, we describe a patient who showed a severe skeletal muscle deficit after the administration of the ALK inhibitor, alectinib, and was successfully treated by corticosteroids without withdrawal from the cancer therapy.

Objective

To assess whether neuropathy with anti-myelin-associated glycoprotein (MAG) antibody may improve after treatment with ibrutinib, an oral inhibitor of Bruton tyrosine kinase, we prospectively treated with ibrutinib a cohort of 3 patients with anti-MAG neuropathy and Waldenström macroglobulinemia (WM).

This paper uses a 3D model for stability assessment of Zhujiabaobao waste dump with ground cracks. The study data were gathered via reconnaissance, geomorphological analysis and laboratory experiment. A 3D finite extended element method model that can consider cracks was then used to calculate the factor of safety (FOS) of the waste dump via the strength reduction technique. The simulation shows the dump to have an FOS of 1.22 and both the position and depth of penetration of cracks in the waste dump have a crucial impact on the stability of the slope. Because the study area is located in a seismically active area, simulation and analysis of the dynamic response of the waste dump under different magnitudes of seismic waves (peak acceleration is 0.05, 0.15, 0.25 and 0.45g) were performed via an explicit dynamic model. The simulation shows that high steps in the slope are particularly responsive to earthquakes. The approach used here for analysing stability under static and dynamic loads is useful for hazard prevention and mitigation.

The decline or drying up of groundwater sources near a tunnel route is damaging to groundwater users. Therefore, forecasting the impact of a tunnel on nearby groundwater sources is a challenging task in tunnel design. In this study, numerical and analytical approaches were applied to the Qomroud water conveyance tunnel (located in Lorestan province, Iran) to assess the impact of tunnelling on the nearby extraction water wells. Using simulation of groundwater-level fluctuation owing to tunnelling, the drawdown at the well locations was determined. From the drawdowns and using Dupuit's equation, the depletion of well flow rates after tunnelling was estimated. To evaluate the results, observed well flow rates before and after tunnelling were compared with the predicted flow rates. The observed and estimated water well flows (before and after tunnelling) showed a regression factor of 0.64, pointing to satisfactory results

The need to characterize and distinguish geographically adjacent aggregate quarry sources prompted the SEM-EDS analysis of pyrite (FeS₂) within fill material taken from eight different quarry sources. This experiment was undertaken to investigate the possibility of geochemically separating these quarry sources based on the major element concentration of their pyrite. The results show that median values for Fe and S vary by up to 7.6 and 8.55 wt% respectively. By implementing statistical methods, including k-means clustering and principal component analysis, it is possible to geochemically discriminate three of the eight sources.

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.

Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d’Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although current serological and molecular detection tools allow a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to assess their susceptibility to antibiotics and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers, as it requires biosafety level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high-content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically designed algorithms targeting cytopathic effects. This method was more efficient than the shell vial assay, at the level of time dependency, when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% versus 78% for shell vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% versus 5% for shell vial), for which most strains were recovered more rapidly with the new technique. In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell vial culture and quantitative PCR.

Quantitative bacterial culture of bronchoalveolar lavage fluids (BALF) is labor-intensive, and the delay involved in performing culture, definitive identification, and susceptibility testing often results in prolonged use of broad-spectrum antibiotics. The Unyvero lower respiratory tract (LRT) panel (Curetis, Holzgerlingen, Germany) allows the multiplexed rapid detection and identification of 20 potential etiologic agents of pneumonia within 5 h of collection. In addition, the assay includes detection of gene sequences that confer antimicrobial resistance. We retrospectively compared the performance of the molecular panel to routine quantitative bacterial culture methods on remnant BALF. Upon testing 175 BALF, we were able to analyze positive agreement of 181 targets from 129 samples, and 46 samples were negative. The positive percent agreement (PPA) among the microbial targets was 96.5%, and the negative percent agreement (NPA) was 99.6%. The targets with a PPA of <100% were Staphylococcus aureus (34/37 [91.9%]), Streptococcus pneumoniae (10/11 [90.9%]), and Enterobacter cloacae complex (2/4 [50%]). For the analyzable resistance targets, concordance with phenotypic susceptibility testing was 79% (14/18). This study found the Unyvero LRT panel largely concordant with culture results; however, no outcome or clinical impact studies were performed.

Screening for Chlamydia trachomatis and Neisseria gonorrhoeae at the pharyngeal, urogenital, and anorectal sites is recommended for men who have sex with men (MSM). Combining the three individual-site samples into a single pooled sample could result in significant cost savings, provided there is no significant sensitivity reduction. The aim of this study was to examine the sensitivity of pooled samples for detecting chlamydia and gonorrhea in asymptomatic MSM using a nucleic acid amplification test. Asymptomatic MSM who tested positive for chlamydia or gonorrhoea were invited to participate. Paired samples were obtained from participants prior to administration of treatment. To form the pooled sample, the anorectal swab was agitated in the urine specimen transport tube and then discarded. The pharyngeal swab and 2 ml of urine sample were then added to the tube. The difference in sensitivity between testing of pooled samples and individual-site testing was calculated against an expanded gold standard, where an individual is considered positive if either pooled-sample or individual-site testing returns a positive result. All samples were tested using the Aptima Combo 2 assay. A total of 162 MSM were enrolled in the study. Sensitivities of pooled-sample testing were 86% (94/109; 95% confidence interval [CI], 79 to 92%]) for chlamydia and 91% (73/80; 95% CI, 83 to 96%) for gonorrhea. The sensitivity reduction was significant for chlamydia (P = 0.02) but not for gonorrhea (P = 0.34). Pooling caused 22 infections (15 chlamydia and 7 gonorrhoea) to be missed, and the majority were single-site infections (19/22). Pooling urogenital and extragenital samples from asymptomatic MSM reduced the sensitivity of detection by approximately 10% for chlamydia but not for gonorrhea.

Microscopy and rapid diagnostic tests (RDTs) are the main diagnostic tools for malaria but fail to detect low-density parasitemias that are important for maintaining malaria transmission. To complement existing diagnostic methods, an isothermal reverse transcription-recombinase polymerase amplification and lateral flow assay (RT-RPA) was developed. We compared the performance with that of ultrasensitive reverse transcription-quantitative PCR (uRT-qPCR) using nucleic acid extracts from blood samples (n = 114) obtained after standardized controlled human malaria infection (CHMI) with Plasmodium falciparum sporozoites. As a preliminary investigation, we also sampled asymptomatic individuals (n = 28) in an area of malaria endemicity (Lambaréné, Gabon) to validate RT-RPA and assess its performance with unprocessed blood samples (dbRT-RPA). In 114 samples analyzed from CHMI trials, the positive percent agreement to uRT-qPCR was 90% (95% confidence interval [CI], 80 to 96). The negative percent agreement was 100% (95% CI, 92 to 100). The lower limit of detection was 64 parasites/ml. In Gabon, RT-RPA was 100% accurate with asymptomatic volunteers (n = 28), while simplified dbRT-RPA showed 89% accuracy. In a subgroup analysis, RT-RPA detected 9/10 RT-qPCR-positive samples, while loop-mediated isothermal amplification (LAMP) detected 2/10. RT-RPA is a reliable diagnostic test for asymptomatic low-density infections. It is particularly useful in settings where uRT-qPCR is difficult to implement.

Lyme borreliosis is a tick-borne disease caused by the Borrelia burgdorferi sensu lato complex. Bio-Rad Laboratories has developed a fully automated multiplex bead-based assay for the detection of IgM and IgG antibodies to B. burgdorferi. The BioPlex 2200 Lyme Total assay exhibits an improved rate of seropositivity in patients with early Lyme infection. Asymptomatic subjects from endemic and nonendemic origins demonstrated a seroreactivity rate of approximately 4% that was similar to other commercial assays evaluated in this study. Coupled to this result was the observation that the Lyme Total assay retained a high first-tier specificity of 96% while demonstrating a relatively high sensitivity of 91% among a well-characterized CDC Premarketing Lyme serum panel. The Lyme Total assay also performs well under a modified two-tier algorithm (sensitivity, 84.4 to 88.9%; specificity, 98.4 to 99.5%). Furthermore, the new assay is able to readily detect early Lyme infection in patient samples from outside North America.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum. The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

The number of onsite clinical microbiology laboratories in hospitals is decreasing, likely related to the business model for laboratory consolidation and labor shortages, and this impacts a variety of clinical practices, including that of banking isolates for clinical or epidemiologic purposes. To determine the impact of these trends, infectious disease (ID) physicians were surveyed regarding their perceptions of offsite services. Clinical microbiology practices for retention of clinical isolates for future use were also determined. Surveys were sent to members of the Infectious Diseases Society of America’s (IDSA) Emerging Infections Network (EIN). The EIN is a sentinel network of ID physicians who care for adult and/or pediatric patients in North America and who are members of IDSA. The response rate was 763 (45%) of 1,680 potential respondents. Five hundred forty (81%) respondents reported interacting with the clinical microbiology laboratory. Eighty-six percent of respondents thought an onsite laboratory very important for timely diagnostic reporting and ongoing communication with the clinical microbiologist. Thirty-five percent practiced in institutions where the core microbiology laboratory has been moved offsite, and an additional 7% (n = 38) reported that movement of core laboratory functions offsite was being considered. The respondents reported that only 24% of laboratories banked all isolates, with the majority saving isolates for less than 30 days. Based on these results, the trend toward centralized core laboratories negatively impacts the practice of ID physicians, potentially delays effective implementation of prompt and targeted care for patients with serious infections, and similarly adversely impacts infection control epidemiologic investigations.

This minireview focuses on the microbiologic evaluation of patients with asymptomatic bacteriuria, as well as indications for antibiotic treatment. Asymptomatic bacteriuria is defined as two consecutive voided specimens (preferably within 2 weeks) with the same bacterial species, isolated in quantitative counts of ≥10⁵ CFU/ml in women, including pregnant women; a single voided urine specimen with one bacterial species isolated in a quantitative count ≥10⁵ CFU/ml in men; and a single catheterized urine specimen with one or more bacterial species isolated in a quantitative count of ≥10⁵ CFU/ml in either women or men (or ≥10² CFU/ml of a single bacterial species from a single catheterized urine specimen). Any urine specimen with ≥10⁴ CFU/ml group B Streptococcus is significant for asymptomatic bacteriuria in a pregnant woman. Asymptomatic bacteriuria occurs, irrespective of pyuria, in the absence of signs or symptoms of a urinary tract infection. The two groups with the best evidence of adverse outcomes in the setting of untreated asymptomatic bacteriuria include pregnant women and patients who undergo urologic procedures with risk of mucosal injury. Screening and treatment of asymptomatic bacteriuria is not recommended in the following patient populations: pediatric patients, healthy nonpregnant women, older patients in the inpatient or outpatient setting, diabetic patients, patients with an indwelling urethral catheter, patients with impaired voiding following spinal cord injury, patients undergoing nonurologic surgeries, and nonrenal solid-organ transplant recipients. Renal transplant recipients beyond 1 month posttransplant should not undergo screening and treatment for asymptomatic bacteriuria. There is insufficient evidence to recommend for or against screening of renal transplant recipients within 1 month, patients with high-risk neutropenia, or patients with indwelling catheters at the time of catheter removal. Unwarranted antibiotics place patients at increased risk of adverse effects (including Clostridioides difficile diarrhea) and contribute to antibiotic resistance. Methods to reduce unnecessary screening for and treatment of asymptomatic bacteriuria aid in antibiotic stewardship.

On 31 December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated coronavirus disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro (1.8 50% tissue culture infective doses [TCID₅₀]/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an additional 42 RdRp-P2-negative specimens (119/273 [43.6%] versus 77/273 [28.2%]; P < 0.001), including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens. The mean viral load of these specimens was 3.21 x 10⁴ RNA copies/ml (range, 2.21 x 10² to 4.71 x 10⁵ RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis of COVID-19.

The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts encoding human leukocyte antigen (HLA) receptors as well as B cell and T cell receptors (BCR and TCR). Determining which alleles an individual possesses for each HLA gene (high-resolution HLA typing) is essential to establish donor–recipient compatibility in organ and bone marrow transplantations. In turn, the repertoires of millions of unique BCR and TCR transcripts in each individual carry a vast amount of health-relevant information. Both short-read RNA-seq-based HLA typing and BCR/TCR repertoire sequencing (AIRR-seq) currently rely on our incomplete knowledge of the genetic diversity at HLA and BCR/TCR loci. Here, we generated over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9% using our nanopore sequencing-based Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol. We used this data set to (1) show that deep and accurate full-length cDNA sequencing can be used to provide isoform-level transcriptome analysis for more than 9000 loci, (2) generate accurate sequences of HLA alleles, and (3) extract detailed AIRR data for the analysis of the adaptive immune system. The HLA and AIRR analysis approaches we introduce here are untargeted and therefore do not require prior knowledge of the composition or genetic diversity of HLA and BCR/TCR loci.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and EGFR. These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in noncancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation, and Rab GTPase regulation was performed using super-resolution and confocal microscopy followed by 3D rendering. MDA-MB-231 breast cancer cells have fewer, but larger EEA1-positive early endosomes compared with MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A overexpression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knockdown of Rab4A lead to increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted in enhanced EGF-induced activation of EGFR-p1068 in MDA-MB-231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation.

We recently reported that restoring the CYP27A1–27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti–prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6–JAK–STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6–JAK–STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation.

Pre-eclampsia (PE)-induced fetal programming predisposes offspring to health hazards in adult life. Here, we tested the hypothesis that pre-eclamptic fetal programming elicits sexually dimorphic inflammatory and cardiovascular complications to endotoxemia in adult rat offspring. PE was induced by oral administration of L-NAME (50 mg/kg per day for seven consecutive days) starting from day 14 of conception. Cardiovascular studies were performed in conscious adult male and female offspring preinstrumented with femoral indwelling catheters. Compared with non-PE male counterparts, intravenous administration of lipopolysaccharide (LPS, 5 mg/kg) to PE male offspring caused significantly greater 1) falls in blood pressure, 2) increases in heart rate, 3) rises in arterial dP/dtmax, a correlate of left ventricular contractility, and 4) decreases in time- and frequency-domain indices of heart rate variability (HRV). By contrast, the hypotensive and tachycardic actions of LPS in female offspring were independent of the pre-eclamptic state and no clear changes in HRV or dP/dtmax were noted. Measurement of arterial baroreflex activity by vasoactive method revealed no sex specificity in baroreflex dysfunction induced by LPS. Immunohistochemical studies showed increased protein expression of toll-like receptor 4 in heart as well as in brainstem neuronal pools of the nucleus of solitary tract and rostral ventrolateral medulla in endotoxic PE male, but not female, offspring. Enhanced myocardial, but not neuronal, expression of monocyte chemoattractant protein-1 was also demonstrated in LPS-treated male offspring. Together, pre-eclamptic fetal programming aggravates endotoxic manifestations of hypotension and autonomic dysfunction in male offspring via exacerbating myocardial and neuromedullary inflammatory pathways.

Dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of type 2 diabetes and obesity because of their beneficial effects on body weight, blood glucose, insulin sensitivity, and food preference, at least short-term. DACRAs activate the receptors for a prolonged time period, resulting in metabolic effects superior to those of amylin. Because of the prolonged receptor activation, different dosing intervals and, hence, less frequent receptor activation might change the efficacy of DACRA treatment in terms of weight loss and food preference. In this study, we compared daily dosing to dosing every other day with the aim of understanding the optimal balance between efficacy and tolerability. Obese and lean male Sprague-Dawley rats were treated with the DACRA KBP-088, applying two different dosing intervals (1.5 nmol/kg once daily and 3 nmol/kg every other day) to assess the effect on body weight, food intake, glucose tolerance, and food preference when given the choice between chow (13% fat) and a high-fat diet (60% fat). Treatment with KBP-088 induced significant weight loss, reduction in adiposity, improvement in glucose control, and altered food preference toward food that is less calorie-dense. KBP-088 dosed every other day (3 nmol/kg) was superior to KBP-088 once daily (1.5 nmol/kg) in terms of weight loss and improvement of food preference. The beneficial effects were evident in both lean and obese rats. Hence, dosing KBP-088 every other day positively affects overall efficacy on metabolic parameters regardless of the lean/obese state, suggesting that less-frequent dosing with KBP-088 could be feasible.

Transient receptor potential (TRP) melastatin 8 (TRPM8) is a temperature-sensing ion channel mainly expressed in primary sensory neurons (A-fibers and C-fibers in the dorsal root ganglion). In this report, we characterized KPR-5714 (N-[(R)-3,3-difluoro-4-hydroxy-1-(2H-1,2,3-triazol-2-yl)butan-2-yl]-3-fluoro-2-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzamide), a novel and selective TRPM8 antagonist, to assess its therapeutic potential against frequent urination in rat models with overactive bladder (OAB). In calcium influx assays with HEK293T cells transiently expressing various TRP channels, KPR-5714 showed a potent TRPM8 antagonistic effect and high selectivity against other TRP channels. Intravenously administered KPR-5714 inhibited the hyperactivity of mechanosensitive C-fibers of bladder afferents and dose-dependently increased the intercontraction interval shortened by intravesical instillation of acetic acid in anesthetized rats. Furthermore, we examined the effects of KPR-5714 on voiding behavior in conscious rats with cerebral infarction and in those exposed to cold in metabolic cage experiments. Cerebral infarction and cold exposure induced a significant decrease in the mean voided volume and increase in voiding frequency in rats. Orally administered KPR-5714 dose-dependently increased the mean voided volume and decreased voiding frequency without affecting total voided volume in these models. This study demonstrates that KPR-5714 improves OAB in three different models by inhibiting exaggerated activity of mechanosensitive bladder C-fibers and suggests that KPR-5714 may provide a new and useful approach to the treatment of OAB.

Behavioral flexibility is important in a changing environment. Previous research suggests that systems consolidation, a long-term poststorage process that alters memory traces, may reduce behavioral flexibility. However, exactly how systems consolidation affects flexibility is unknown. Here, we tested how systems consolidation affects: (1) flexibility in response to value changes and (2) flexibility in response to changes in the optimal sequence of actions. Mice were trained to obtain food rewards in a Y-maze by switching nose pokes between three arms. During initial training, all arms were rewarded and mice simply had to switch arms in order to maximize rewards. Then, after either a 1 or 28 d delay, we either devalued one arm, or we reinforced a specific sequence of pokes. We found that after a 1 d delay mice adapted relatively easily to the changes. In contrast, mice given a 28 d delay struggled to adapt, especially for changes to the optimal sequence of actions. Immediate early gene imaging suggested that the 28 d mice were less reliant on their hippocampus and more reliant on their medial prefrontal cortex. These data suggest that systems consolidation reduces behavioral flexibility, particularly for changes to the optimal sequence of actions.

In obsessive–compulsive disorder (OCD), functional behaviors such as checking that a door is locked become dysfunctional, maladaptive, and debilitating. However, it is currently unknown how aversive and appetitive motivations interact to produce functional and dysfunctional behavior in OCD. Here we show a double dissociation in the effects of anxiogenic cues and sensitivity to rewarding stimuli on the propensity to develop functional and dysfunctional checking behavior in a rodent analog of OCD, the observing response task (ORT). While anxiogenic manipulations of perceived threat (presentation of threat-associated contextual cues) and actual threat (punishment of incorrect responding on the ORT) enhanced functional checking, dysfunctional checking was unaffected. In contrast, rats that had previously been identified as "sign-trackers" on an autoshaping task—and therefore were highly sensitive to the incentive salience of appetitive environmental cues—selectively showed elevated levels of dysfunctional checking under a range of conditions, but particularly so under conditions of uncertainty. These data indicate that functional and dysfunctional checking are dissociable and supported by aversive and appetitive motivational processes, respectively. While functional checking is modulated by perceived and actual threat, dysfunctional checking recruits appetitive motivational processes, possibly akin to the "incentive habits" that contribute to drug-seeking in addiction.

We investigated whether cycloheximide (CHX) would induce amnesia for the stress-induced impairment of extinction retrieval. First, a single restraint stress session was demonstrated to impair extinction retrieval, but not fear conditioning. A second experiment showed that when CHX was administered immediately after restraint, rats exhibited significant extinction retrieval at test (i.e., retrograde amnesia for the stress). In a third experiment, the stress session impaired various amounts of extinction durations, suggesting that the stress inhibited extinction retrieval rather than enhancing the original fear learning. These results suggest memories for acute stress are susceptible to disruption, which could have clinical implications.

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.

When we critically assess the reason for the current dominance of ⁶⁸Ga-labeled peptides and peptide-like ligands in radiopharmacy and nuclear medicine, we have to conclude that the major advantage of such radiopharmaceuticals is the apparent lack of suitable ¹⁸F-labeling technologies with proven clinical relevance. To prepare and to subsequently perform a clinical proof-of-concept study on the general suitability of silicon-fluoride-acceptor (SiFA)–conjugated radiopharmaceuticals, we developed inhibitors of the prostate-specific membrane antigen (PSMA) that are labeled by isotopic exchange (IE). To compensate for the pronounced lipophilicity of the SiFA unit, we used metal chelates, conjugated in close proximity to SiFA. Six different radiohybrid PSMA ligands (rhPSMA ligands) were evaluated and compared with the commonly used ¹⁸F-PSMA inhibitors ¹⁸F-DCFPyL and ¹⁸F-PSMA-1007. Methods: All inhibitors were synthesized by solid-phase peptide synthesis. Human serum albumin binding was measured by affinity high-performance liquid chromatography, whereas the lipophilicity of each tracer was determined by the n-octanol/buffer method. In vitro studies (IC₅₀, internalization) were performed on LNCaP cells. Biodistribution studies were conducted on LNCaP tumor–bearing male CB-17 SCID mice. Results: On the laboratory scale (starting activities, 0.2–9.0 GBq), labeling of ¹⁸F-rhPSMA-5 to -10 by IE was completed in < 20 min (radiochemical yields, 58% ± 9%; radiochemical purity, >97%) with molar activities of 12–60 GBq/μmol. All rhPSMAs showed low nanomolar affinity and high internalization by PSMA-expressing cells when compared with the reference radiopharmaceuticals, medium-to-low lipophilicity, and high human serum albumin binding. Biodistribution studies in LNCaP tumor–bearing mice revealed high tumor uptake, sufficiently fast clearance kinetics from blood, low hepatobiliary excretion, fast renal excretion, and very low uptake of ¹⁸F activity in bone. Conclusion: The novel ¹⁸F-rhPSMA radiopharmaceuticals developed under the radiohybrid concept show equal or better targeting characteristics than the established ¹⁸F-PSMA tracers ¹⁸F-DCFPyL and ¹⁸F-PSMA-1007. The unparalleled simplicity of production, the possibility to produce the identical ⁶⁸Ga-labeled ¹⁹F-⁶⁸Ga-rhPSMA tracers, and the possibility to extend this concept to true theranostic radiohybrid radiopharmaceuticals, such as F-Lu-rhPSMA, are unique features of these radiopharmaceuticals.

¹⁸F-prostate-specific membrane antigen (PSMA)-1007 is excreted mainly through the liver. We benchmarked the performance of ¹⁸F-PSMA-1007 against 3 renally excreted PSMA tracers. Methods: Among 668 patients, we selected 27 in whom PET/CT results obtained with ⁶⁸Ga-PSMA-11, ¹⁸F-DCFPyL (2-(3-(1-carboxy-5-[(6-[¹⁸F]fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentanedioic acid), or ¹⁸F-JK-PSMA-7 (JK, Juelich-Koeln) were interpreted as equivocal or negative or as oligometastatic disease (PET-1). Within 3 wk, a second PET scan with ¹⁸F-PSMA-1007 was performed (PET-2). The confidence in the interpretation of PSMA-positive locoregional findings was scored on a 5-point scale, first in routine diagnostics (reader 1) and then by an independent second evaluation (reader 2). Discordant PSMA-positive skeletal findings were examined by contrast-enhanced MRI. Results: For both readers, ¹⁸F-PSMA-1007 facilitated the interpretability of 27 locoregional lesions. In PET-2, the clinical readout led to a significantly lower number of equivocal locoregional lesions (P = 0.024), and reader 2 reported a significantly higher rate of suspected lesions that were falsely interpreted as probably benign in PET-1 (P = 0.023). Exclusively in PET-2, we observed a total of 15 PSMA-positive spots in the bone marrow of 6 patients (22%). None of the 15 discordant spots had a morphologic correlate on the corresponding CT scan or on the subsequent MRI scan. Thus, ¹⁸F-PSMA-1007 exhibits a significantly higher rate of unspecific medullary spots (P = 0.0006). Conclusion: ¹⁸F-PSMA-1007 may increase confidence in interpreting small locoregional lesions adjacent to the urinary tract but may decrease the interpretability of skeletal lesions.

Monoclonal antibodies (mAbs) against programmed cell death 1 (PD-1), such as nivolumab and pembrolizumab, are associated with high response rates in patients with relapsed or refractory classic Hodgkin lymphoma (HL). To date, no prognostic factor for overall survival (OS) has been established with these agents in HL. We examined whether the first early response assessment evaluated using ¹⁸F-FDG PET/CT may be associated with OS in this setting. Methods: This retrospective study included 45 patients from 34 institutions. In a masked, centralized review, 3 independent radiologists classified PET/CT scans obtained at a median of 2.0 mo (interquartile range, 1.7–3.7 mo) after nivolumab initiation using existing criteria (i.e., 2014 Lugano classification and 2016 LYRIC). Patients were classified according to 4 possible response categories: complete metabolic response (CMR), partial metabolic response (PMR), no metabolic response (NMR), or progressive metabolic disease (PMD). Because the OS of patients with NMR and PMR was similar, they were grouped together. OS was estimated using the Kaplan–Meier method and compared between groups using log-rank testing. Results: Eleven patients (24%) died after a median follow-up of 21.2 mo. The classification was identical between Lugano and LYRIC because all 16 progression events classified as indeterminate response per LYRIC were confirmed on subsequent evaluations. Both Lugano and LYRIC classified patients as CMR in 13 cases (29%), PMD in 16 (36%), NMR in 4 (9%), and PMR in 12 (27%). The 2-y OS probability was significantly different in patients with PMD (0.53; 95% confidence interval [95%CI], 0.32–0.87), NMR or PMR (0.80; 95%CI, 0.63–1.00), and CMR (1.00; 95%CI, 1.00–1.00) in the overall population (P = 0.02, 45 patients), as well as according to a landmark analysis at 3 mo (P = 0.05, 32 patients). Conclusion: In relapsed or refractory HL patients treated with anti-PD-1 mAbs, the first early PET/CT assessment using either Lugano or LYRIC predicted OS and allowed early risk stratification, suggesting that PET/CT might be used to develop risk-adapted strategies.

Infection with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may remain asymptomatic, leading to under-recognition of the related disease, coronavirus disease, 2019 (COVID-19), and to incidental findings in nuclear imaging procedures performed for standard clinical indications. Here, we report about our local experience in a region with high COVID-19 prevalence and dynamically increasing infection rates. Methods: Within the 8-d period of March 16–24, 2020, hybrid imaging studies of asymptomatic patients who underwent ¹⁸F-FDG PET/CT or ¹³¹I SPECT/CT for standard oncologic indications at our institution in Brescia, Italy, were analyzed for findings suggestive of COVID-19. The presence, radiologic features, and metabolic activity of interstitial pneumonia were identified, correlated with the subsequent short-term clinical course, and described in a case series. Results: Six of 65 patients (9%) who underwent PET/CT for various malignancies showed unexpected signs of interstitial pneumonia on CT and elevated regional ¹⁸F-FDG avidity. Additionally, 1 of 12 patients who received radioiodine for differentiated thyroid carcinoma also showed interstitial pneumonia on SPECT/CT. Five of 7 patients had subsequent proof of COVID-19 by reverse-transcriptase polymerase chain reaction. The remaining 2 patients were not tested immediately but underwent quarantine and careful monitoring. Conclusion: Incidental findings suggestive of COVID-19 may not be infrequent in hybrid imaging of asymptomatic patients in regions with an expansive spread of SARS-CoV-2. Nuclear medicine services should prepare accordingly.

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs’ role in localizing the BLM–TOP3A–RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

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).

A multidisciplinary endocrinologist-led shared medical appointment (SMA) model showed statistically significant reductions in A1C from baseline over 3 years that were not significantly different from appointments with endocrinologists or primary care providers alone within a resource-poor population. Similarly, the SMA model achieved clinical outcomes on par with endocrinologist-only visits with the added benefit of improving endocrine provider productivity and specialty access for patients. Greater patient engagement with the SMA model was associated with significantly lower A1C.

Background

The increasing incidence of life-threatening Pneumocystis pneumonia (PCP) in non-HIV immunocompromised patients is a global concern. Yet, no reports have examined the prognostic significance of pre-existing interstitial lung disease (ILD) in non-HIV PCP.

Background

COPD patients account for a large proportion of lung transplants; lung transplantation survival benefit for COPD patients is not well established.

Introduction

Immunotherapy has become the standard of care in advanced non-small cell lung cancer (NSCLC). We aimed to quantify the economic impact, in France, of anti-PD-1 therapy for NSCLC.

ABSTRACT

The chemical structures of soluble fiber carbohydrates vary from source to source due to numerous possible linkage configurations among monomers. However, it has not been elucidated whether subtle structural variations might impact soluble fiber fermentation by colonic microbiota. In this study, we tested the hypothesis that subtle structural variations in a soluble polysaccharide govern the community structure and metabolic output of fermenting microbiota. We performed in vitro fecal fermentation studies using arabinoxylans (AXs) from different classes of wheat (hard red spring [AX_HRS], hard red winter [AX_HRW], and spring red winter [AX_SRW]) with identical initial microbiota. Carbohydrate analyses revealed that AX_SRW was characterized by a significantly shorter backbone and increased branching compared with those of the hard varieties. Amplicon sequencing demonstrated that fermentation of AX_SRW resulted in a distinct community structure of significantly higher richness and evenness than those of hard-AX-fermenting cultures. AX_SRW favored OTUs within Bacteroides, whereas AX_HRW and AX_HRS favored Prevotella. Accordingly, metabolic output varied between hard and soft varieties; higher propionate production was observed with AX_SRW and higher butyrate and acetate with AX_HRW and AX_HRS. This study showed that subtle changes in the structure of a dietary fiber may strongly influence the composition and function of colonic microbiota, further suggesting that physiological functions of dietary fibers are highly structure dependent. Thus, studies focusing on interactions among dietary fiber, gut microbiota, and health outcomes should better characterize the structures of the carbohydrates employed.

IMPORTANCE Diet, especially with respect to consumption of dietary fibers, is well recognized as one of the most important factors shaping the colonic microbiota composition. Accordingly, many studies have been conducted to explore dietary fiber types that could predictably manipulate the colonic microbiota for improved health. However, the majority of these studies underappreciate the vastness of fiber structures in terms of their microbial utilization and omit detailed carbohydrate structural analysis. In some cases, this causes conflicting results to arise between studies using (theoretically) the same fibers. In this investigation, by performing in vitro fecal fermentation studies using bran arabinoxylans obtained from different classes of wheat, we showed that even subtle changes in the structure of a dietary fiber result in divergent microbial communities and metabolic outputs. This underscores the need for much higher structural resolution in studies investigating interactions of dietary fibers with gut microbiota, both in vitro and in vivo.