Delwest is bringing some income-restricted apartments to an “impressive” location.

Center for Biological Diversity is suing sued the state health department over two Crestone Peak sites.

Gambling, Risky Pranks and Lucrative Contracts: Inside the Streaming Site Kick

There are many new mediation syllabi on the DRLE website. There now are two pages for these syllabi.  One page is for courses focused on the perspective of neutrals and the other focused on the perspective of representatives in mediation.  Both webpages have separate sections for simulation and clinical courses. The webpages include a lot … Continue reading New Mediation Syllabi on DRLE Website →

I've kind of been ignoring this blog for a while. That's nothing new... there have been multi-year periods with no posts. I'm still writing stuff...

Мой сайт временно не открывается из России у большинства провайдеров (у меня из Питера открывается). И это нормально. Мы этого ждали, мы с вами к этому давно готовы. Но спешу успокоить: связано это вовсе не с моим скромным сайтом. Он не под запретом и не под санкциями. Связано это с тем, что он, как сотни тысяч других сайтов, проксируется через CloudFlare. CloudFlare — крупнейший мировой сервис (ему, например, принадлежит IP 1.1.1.1), который, скажем так, сильно помогает владельцу строить доступ к своему сайту. С недавнего времени там используется новая технология соединения под названием ECH (Encrypted Client Hello) на TLS версии 1.3, и вот ее Роскомнадзор принялся блокировать. Простыми словами: когда вы открываете ссылку https (вместо старого протокола http), то ваше соединение шифрованное. И Роскомнадзор не видит, что за данные вы читаете-передаете. Он просто видит, что идет обмен какими-то зашифрованными данными с сайтом по имени lleo.me Это Роскомнадзор сильно огорчает. https именно для этого и придумали ещё в прошлом веке. Но раньше Роскомнадзору было хотя бы видно имя сайта. В новом протоколе обмена имя сайта тоже не видно. Пользователь пошел куда-то и прочитал там три килобайта чего-то. Это совсем расстраивает Роскомнадзор. Поэтому пару дней назад он начал тупо блокировать протокол ECH, как минимум — для CloudFlare. Не блокируются только заходы с браузеров, которые не поддерживают TLS 1.3 (например, wget). Думаю, теоретически посетитель может отключить у себя в браузере TLS версии 1.3, но я не пробовал и вам категорически не советую — много чего может рухнуть. А советую раздобыть уже наконец VPN. Владельцы сайтов могут это поправить на своей стороне: в своих настройках на Сloudflare выключить ECH — но только если вы платный абонент CloudFlare. Зато бесплатные владельцы сайтов могут в настройках CloudFlare просто выключить TLS 1.3 в разделе «SSL/TLS» / «Edge Certificates» / «TLS 1.3», и будет тот же эффект. Однако я не побегу это делать сразу. Почему? У меня в ближайшие дни до 12 числа адова дохерища работы и все равно нет времени писать новые посты в дневник. Но и чисто из педагогических соображений спешить не следует. Во-первых, VPN должен стать таким же элементом личной гигиены как мыло в каждом доме. Каждая новая проблема, созданная Роскомнадзором, должна лишь напоминать об этом. Во-вторых, говноеды Роскомнадзора должны сперва вдоволь наесться говна. Ведь через CloudFlare завязан не какой-то там никчемный lleo.me, а сотни тысяч сайтов, в том числе критически важные: заводы, магазины, поликлиники, детские садики, билетные кассы, и всё это точно так же наебнулось. Так что теоретически даже есть мизерный шанс, что фраер сдаст назад. В общем, если у вас действительно проблемы с моим сайтом, просто советую недельку подождать и не заходить. Я постараюсь, чтобы до 13 ноября здесь не было ничего, достойного вашего внимания. А 13 ноября мы что-нибудь с этим придумаем, и я новый рассказ, например, выложу.

If you’re planning on creating and launching a new website you should immediately be thinking about how to market your new website on the internet before having it designed. There are a ton of great web design companies available who can provide you with a great design and offer free consultations. One in particular I […]

As you may know or not know, Google is not providing keywords data when users are logged in to their accounts. This has been an on-going topic throughout the SEO world for sometime now. Link Worx Seo wrote about this topic on 10/23/2011 and 11/03/2011 and the effects it is having on SEO analysis. As […]

Overall Added Value As a Web Designer, Programmer and SEO Specialist it all goes hand and hand for the most part. Knowing your target country or audience can aid in the delivery of your message and what you are trying to accomplish. The design and layout are for sure areas that go overlooked and under […]

Why do you Place Social Buttons on a Website? During a discussion on LinkedIn, a gentleman posed the question about not being able to understand the reason social media buttons are on main business websites. I thought it might be nice to cover some of the main purposes of social media strategies and what can […]

This is some information I compiled surrounding domain names being purchased or expiring. I came across this on Google while doing some research on domain names and link building. Domain names that are expired are given a page rank (PR) of zero once they are expired. Now there are some time stipulations set on domain […]

What has the Agenda Been Like Okay, recently there have been significant changes going on behind the scenes for the company. Not only has the company created new added features to the custom content management system, a new invoice system has been created as well. The new editions to the system required some intensive Cold […]

Our Blogging Delay Update First off, if anyone who is a subscriber has been wondering why the news letter has been the same for about two months now, it has been because of being extremely busy. There has been an extensive amount of work being completed elsewhere for another company over the last few months. […]

Free Sitemap Creation Considering all the available resources for creating sitemaps, this one is the best around as far as I am concerned. I personally have tinkered around with several sitemap programs and multiple sitemap generators online. Some charge for these services but there are plenty of free resources available online. The option is yours. […]

Directory Inclusion Service : A large percentage of the world is using the internet for a variety of thing’s nowadays. Because of this a large number of directories have been created in effort to generate revenue and localization for websites to be categorized into easily searchable databases that list different websites. Search engines have made […]

Well, let me start off by saying thing’s have been busy since the last post back in early November. Most of my time and focus has been spent updating websites to HTML5 code. So my apologize for the long delay of writing. There is still plenty of updating to complete so it could be a […]

Okay, recently there has been some discussion surrounding the Google Authorship and Publisher topic. So I figured what a great time to cover another article for the topic since it has been part of an update being done for some websites I am working on. First off, there has been some more talk about the […]

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Meeting the challenge to gently place a home into a valley below the access road and adjacent to a scenic river, Weston Residence is both a primary residence and an award-winning example of minimal site impact architectural design. [...]

Show Notes for Episode 4: Subscribe to the Internet Musician Podcast with iTunes: As a musician, you need a good website.  You know that. But you really don’t need to spend years trying to understand the technology. You also don’t want to spend tons of time working on your site without knowing if you’re doing things […]

This month's nycdevops meetup speaker is Nathen Harvey of Google, who will give a talk titled "Introduction to Site Reliability Engineering".

The talk starts at 5pm sharp! (NY is in US/Eastern)

Please RSVP! See you there!

https://www.meetup.com/nycdevops/events/272956481/

(This is a virtual meetup. Everyone around the world is invited!)

Improving organic traffic can sometimes be challenging, but if you apply the correct SEO strategies, the hard work will eventually pay off. Search engine traffic is great because it’s relevant and targeted. For most websites, organic traffic converts much better than traffic from social media or other sources. Another great advantage is that visits from […]

The post 10 Smart Ways To Increase Organic Traffic To A Website appeared first on RSSground.com.

As a small business owner, regardless of what business you are in, you need to have a great...

The post Why It Pays to Invest in your Website Design appeared first on Ignition Media.

If you’re like most business owners, you probably think of your website as an online brochure. It’s a...

The post How to Make Your Website a Lead-Generating Machine appeared first on Ignition Media.

Do you ever sit down to work on your website and wonder what you can do to get...

The post How to Convert Website Visitors Into Paying Customers appeared first on Ignition Media.

Think your website is fine the way it is? Think again. Even if you’re not seeing a decrease...

The post 10 Features Every Great Website Must Have appeared first on Ignition Media.

The website is the foundation of any successful online business. It’s the first impression potential customers have of...

The post 10 Ignition Media Tactics to Take Your Website to the Next Level appeared first on Ignition Media.

At Ignition Media, as we embrace 2024, we’re keenly aware of the evolving trends in web design that...

The post The Top Website Design Trends for 2024 appeared first on Ignition Media.

Maintaining an edge in SEO is crucial in the dynamic world of digital marketing. WordPress SEO plugins play...

The post Top WordPress SEO Plugins for 2024: Enhance Your Website’s Performance appeared first on Ignition Media.

Chikankari specialty Step into a world where every thread tells a story and every pattern sings a story song. Chikankari House stands not only as an advertisement but as the custodian of a centuries-old art form. Traditional Indian embroidery is not only preserved here; It is celebrated, nurtured and evolved. Chikankari with its subtle beauty and intricate details weaves the fabric of India’s rich cultural story. Chikankari House: Carrying forward a valuable asset Chikankari-property History and Origins Chikankari House takes great pride in being the torchbearer of an art form that has deep roots in ancient India. Known for its exquisite beauty and subtlety, Chikankari is often associated with the Nawab’s land in Lucknow. This delicate weaving style is believed to have flourished during the Mughal era under the patronage of Noor Jahan, wife of Emperor Jehangir. Chinkari, however, is more than just embroidery and decoration; It symbolizes an interesting story told through fashion. The story begins in mythology, where Lord Krishna’s wife Radha is said to have expressed her love with unique colorful chikankari patterns embroidered on cloth This Connection imbues chikankari with divine substance and emotional meaning, and owes it and rich textiles of rich cultural heritage again Lucknow is situated. Evolution Over the Years Like a river that changes its course over time yet never abandons its source, Chikankari too went through various transformations over the centuries. Yet, it painstakingly preserved its original essence. Chikankari artisans of the past passed on their art to younger generations, ensuring that it continued with all its skill and fervour. Notable figures in history have contributed to the popularity of this intricate craft over the years. British viceroys, Queen Victoria herself, and more recently, celebrities, and fashion designers have all played crucial roles in keeping Chikankari in the public consciousness. In its nascent stages, Chikankari was typically portrayed in white-on-white embroidery on muslin cloth, epitomizing the essence of understated elegance. Over time, the craft embraced colored threads, sequins, beads, and various fabric types, transforming Chikankari into a versatile and appealing element in modern fashion. Through the eye of the House of Chikankari, this beautiful amalgamation of past and present turns a simple piece of fabric into an alluring canvas. And as the world evolves, so do we, making sure the essence of Chikankari, the legacy of Kesari, continues to enthral generations to come. The Craftsmanship of Chikankari: An Intricate Symphony of Stitches Understanding the Art When it comes to understanding the art of Chikankari, the process begins even before the first stitch is laid. The House of Chikankari starts its crafting journey with the selection of just the right fabric – one that holds onto the charm of the delicate stitches, absorbing the soul of Lucknow into its texture. Traditionally, fabrics like muslin, cotton, and silk have been used owing to their soft texture and durability. The art features multiple unique stitches, each lending a distinctive texture to the piece. One such stitch, traditionally known as the “Bakhia” or the shadow work, stands as the backbone of Chikankari. Symbolic of Chikankari’s subtlety, Bakhia is an intricate technique where the thread’s shadow reflects on the cloth, creating a play of light and shade that brings the fabric to life. The artist behind the craft Behind every piece of Chikankari art from Chikankari House is an artist – patient, dedicated and full of unimaginable skill. They are quiet poets who tell stories through their work, weaving stories of tradition and heritage. Becoming a Chikankari artisan is not a job but a professional journey, often passed down through generations. Their training usually begins at an early age, and they spend years mastering the details – from learning different fabrics to understanding patterns and knots While it’s hard to put a number on the many unsung heroes nurturing this art form, an estimated 2.5 million artists across India are associated with Chikankari, according to the European Center for Development Policy and Management , sources that this art show succeeds on a global stage, spreading the appeal of our cultural heritage far and wide. House of Chikankari Today The House of Chikankari, rooted deeply in traditional artistry, is embracing the winds of change to sail towards modernity with grace and resilience. Today, this time-honored craft marries age-old techniques with contemporary influences, gracefully striding into the global fashion arena. Modern Innovations In the realm of Chikankari, modern technology and evolving fashion sensibilities play pivotal roles in elevating this craft to cater to contemporary tastes without losing its essence. Digital Design and Promotion: Techniques such as digital embroidery design and online marketing are helping Chikankari reach global audiences. The House of Chikankari leverages social media and e-commerce to showcase its intricate designs worldwide. New Materials and Stitches: While preserving the traditional stitches, innovative materials like georgette and net are being used, along with traditional cotton and silk. The inclusion of embellishments such as sequins and beads adds to the allure of modern Chikankari outfits. Fashion Integration: Notable fashion designers and brands, including the House of Chikankari, have been instrumental in incorporating Chikankari into high fashion. Collections showcased at major fashion weeks have featured Chikankari, placing it on the global fashion map. Sustaining the Craft The journey of Chikankari, from the streets of Lucknow to the fashion capitals of the world, has been supported by various efforts to preserve its legacy. Government and NGO Efforts: Initiatives like the Geographical Indications (GI) tag for Lucknow Chikankari by the Indian government have aided in protecting its identity. NGOs and social enterprises collaborate with artisans, providing them with fair wages and better work conditions. Educational Programs: Specialized training programs and workshops aim at skill enhancement and introducing the youth to Chikankari, ensuring the inheritance of this craft by future generations. Exhibitions and Workshops: Events such as the “Chikankari Exhibition” in major cities and the participation of Chikankari craft in international craft fairs increase awareness and appreciation for this delicate embroidery work. Through these efforts, the House of Chikankari not only survives but thrives in the landscape of modern fashion and global markets. The blend of traditional craftsmanship with contemporary innovation ensures that Chikankari remains not just a craft of the past but a living tradition, evolving and growing with each stitch in time. Investing in Chikankari Investing in Chikankari is not just a purchase—it’s an embrace of a heritage, a story woven into fabric. This section provides a comprehensive guide to ensure you select authentic pieces and maintain them, so they last a lifetime. Guide to Buying Chikankari Selecting Authentic Pieces Look for Handcraftsmanship: Authentic Chikankari is hand-embroidered. Look closely at the stitching. Irregularities often indicate handwork, as opposed to the uniform stitches of machine embroidery. Fabric Quality: Traditional Chikankari is done on natural fabrics like cotton, silk, and muslin. A gentle touch and the slightly irregular texture of natural fabrics are good indicators of quality. Design Details: Authentic Chikankari flaunts intricate designs that can range from floral motifs to paisley and geometric patterns. The richness in details often signifies authentic workmanship. Where to Buy: Prefer purchasing from recognized artisans, local markets in Lucknow, or reputable online stores specializing in Chikankari. Some reputable brands connected to the House of Chikankari offer quality assurance. Price Range and Factors Affecting Cost Artisan vs. Mass-Produced: Handcrafted Chikankari pieces, especially those from skilled artisans, will be priced higher than mass-produced ones. The uniqueness and quality justify the cost. Complexity of Work: The more intricate the embroidery and the denser the design, the higher the price. Work that takes longer to produce is naturally more valuable. Fabric Type: The choice of fabric impacts the price. Silk-based Chikankari garments are pricier than those crafted on cotton due to the cost of the material. Caring for Chikankari Garments To ensure your Chikankari pieces remain as timeless as their tradition, following proper care instructions is crucial. Washing Instructions: It’s best to hand wash Chikankari clothes in cold water with a mild detergent. Avoid wringing the fabric harshly. For machine wash, use a gentle cycle. Drying and Ironing: Always dry Chikankari garments in the shade. Direct sunlight can fade the delicate threads. While ironing, place a thin cloth over the garment to prevent direct contact with the iron. Storage: Store Chikankari apparel in a cool, dry place. Use padded hangers for items like sarees and kurtas to avoid creases. For best preservation, wrap the garments in soft, breathable fabric. By following these guidelines, you can invest wisely in Chikankari, ensuring you own a piece of heritage that brings elegance and traditional charm to your wardrobe. This enduring craftsmanship, with the right care, can be part of your legacy, cherished and passed down through generations. Chikankari Future As we navigate the crowded world of fashion and fashion, the House of Chikankari remains open in the face of obstacles and opportunities. Navigating a sea of ​​change, the delicate beauty of embroidery clings to its heritage. Challenges and opportunities Challenges Competition with machine-made products: One of the most important issues is competition from faster and cheaper looms that imitate Chikankari. Preservation of traditional skills: There is a danger that the meticulous skills of Chikankari craftsmen will be lost in today’s fast-paced manufacturing environment. Economic Development: Designers often face financial challenges due to irregular demand for materials and a lot of time spent on crafts. opportunity Design innovation: Innovation that captures the essence of Chikankari while appealing to contemporary aesthetics can create new appeal. Online Marketplace: With the rise of e-commerce, Chikankari offers a global platform, connecting skilled artisans with consumers directly. Sustainable clothing: As consumers become more eco-conscious, demand for sustainable and ethically produced clothing such as chikankari may increase. What is happening in the future Trends in the fashion industry, as well as consumer preferences, point to a different future for Chikankari. There is the possibility that: Fusion Wear: Combine chikankari and western fashion elements for unique outfits. Heritage promotion: The use of chikankari as a contemporary fashion can be a cultural promotion, especially among the youth. Technology integration: Technology can provide immersive shopping experiences such as virtual reality, allowing consumers to interact with artistic origins and stories. conclusion Chikankari House, an Indian heritage fortress plays an important role in traditional handloom weaving. It’s not just historical art preservation; It supports the livelihood of countless artists. Cultural Identity: Supporting Chikankari is like preserving an irreplaceable part of India’s cultural identity. Investing in Heritage: Each purchase of a chikankari is an investment in a tradition that has been handed down for generations. Call to action: Explore the timeless beauty of authentic chikankari embroidery. There is a story embedded in every thread. Sharing history of every purchase. Chikankari House is not just alive; it is adaptive, conquering, and moving forward to meet dog after dog adorned with canine charms. It invites us to be not only spectators but participants in the preservation of this complex art form.

The post House of Chikankari: Unveiling the Secrets Behind Exquisite Embroidery appeared first on Geeky Traveller.

Modern commerce is a highly complex global business that is always evolving – and the pace of change is only accelerating. If you work in digital commerce, it’s crucial to stay up-to-date with the latest developments in technology, business, shopper behavior and trends, laws and regulations, payments, logistics and more. How can you stay on […]

If you run an eCommerce website, you may prefer to use a graphic as the call-to-action button for your subscription-style products or services. Below you can find a number of different subscription buttons suitable for use on a website’s landing page or the product page. Want some Buy Now button or Add to Cart button […]

The post Subscription Button Graphics for eCommerce Websites appeared first on Tips and Tricks HQ.

In this blog post, you can find a range of different ‘Buy Now’ buttons that can be used on a website as the ‘call-to-action’ button for a product or service you are trying to sell. There is a range of different size buttons, in a number of different colors to fit with the aesthetics of […]

The post Buy Now Button Graphics for eCommerce Websites appeared first on Tips and Tricks HQ.

Do you run a charitable website? Or do you offer content free to the public? Perhaps it is time that you add a donation button to start monetizing your website. In this tutorial, we will walk you through the process of adding a PayPal donation button to your WordPress website, and even those without developer […]

The post Accept Donations via PayPal from Your WordPress Site Easily appeared first on Tips and Tricks HQ.

The mobile phone market is teeming with a large number of brands, which have been capable of gaining worldwide recognition. Similar to other, this market is also witnessing fiercely competitive scenario.

Whether America realized it or not, on the last day of Trump's Presidency, Trump was already making his case for his return to the Oval Office. And it was a strong case in his favor.

The post The STRONGEST Argument For Re-Electing Trump Was Published On The White House Website appeared first on Clash Daily.

The main protease (3CL Mpro) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL Mpro possesses an unusual catalytic dyad composed of Cys145 and His41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active-site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL Mpro, which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form in which Cys145 is in the negatively charged thiolate state and His41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen-bonding networks in the active-site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme's electrostatic environment.

Ivan Matic
Jan 1, 2008; 7:132-144
Research

Yi Zhang
Sep 1, 2005; 4:1240-1250
Research

Sebastian A. Wagner
Oct 1, 2011; 10:M111.013284-M111.013284
Research

Yu Xue
Sep 1, 2008; 7:1598-1608
Research

Lys234 is one of the residues present in class A β-lactamases that is under selective pressure due to antibiotic use. Located adjacent to proton shuttle residue Ser130, it is suggested to play a role in proton transfer during catalysis of the antibiotics. The mechanism underpinning how substitutions in this position modulate inhibitor efficiency and substrate specificity leading to drug resistance is unclear. The K234R substitution identified in several inhibitor-resistant β-lactamase variants is associated with decreased potency of the inhibitor clavulanic acid, which is used in combination with amoxicillin to overcome β-lactamase–mediated antibiotic resistance. Here we show that for CTX-M-14 β-lactamase, whereas Lys234 is required for hydrolysis of cephalosporins such as cefotaxime, either lysine or arginine is sufficient for hydrolysis of ampicillin. Further, by determining the acylation and deacylation rates for cefotaxime hydrolysis, we show that both rates are fast, and neither is rate-limiting. The K234R substitution causes a 1500-fold decrease in the cefotaxime acylation rate but a 5-fold increase in kcat for ampicillin, suggesting that the K234R enzyme is a good penicillinase but a poor cephalosporinase due to slow acylation. Structural results suggest that the slow acylation by the K234R enzyme is due to a conformational change in Ser130, and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser130 and such changes drastically reduce cephalosporin but not penicillin hydrolysis, we suggest that clavulanic acid paired with an oxyimino-cephalosporin rather than penicillin would impede the evolution of resistance.

Expert

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3'-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5'-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC50 values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.

The life cycle of malaria parasites in both their mammalian host and mosquito vector consists of multiple developmental stages that ensure proper replication and progeny survival. The transition between these stages is fueled by nutrients scavenged from the host and fed into specialized metabolic pathways of the parasite. One such pathway is used by Plasmodium falciparum, which causes the most severe form of human malaria, to synthesize its major phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Much is known about the enzymes involved in the synthesis of these phospholipids, and recent advances in genetic engineering, single-cell RNA-Seq analyses, and drug screening have provided new perspectives on the importance of some of these enzymes in parasite development and sexual differentiation and have identified targets for the development of new antimalarial drugs. This Minireview focuses on two phospholipid biosynthesis enzymes of P. falciparum that catalyze phosphoethanolamine transmethylation (PfPMT) and phosphatidylserine decarboxylation (PfPSD) during the blood stages of the parasite. We also discuss our current understanding of the biochemical, structural, and biological functions of these enzymes and highlight efforts to use them as antimalarial drug targets.