High-throughput screening of the Saccharomyces cerevisiae genome for 2-amino-3-methylimidazo [4,5-f] quinoline resistance identifies colon cancer-associated genes.

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for 5 or 10 generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. We identified 424 ORFs, including 337 genes of known function, in duplicate screens of the "humanized" collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and "humanized" libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). These studies underscore the role of ribosomal protein genes in conferring IQ resistance, and illuminate DNA repair pathways for conferring resistance to activated IQ.

Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Mechanistic modeling, simulation, and optimization of mixed-mode chromatography for an antibody polishing step.

Mixed-mode chromatography combines features of ion-exchange chromatography and hydrophobic interaction chromatography and is increasingly used in antibody purification. As a replacement for flow-through operations on traditional unmixed resins or as a pH-controlled bind-and-elute step, the use of both interaction modes promises a better removal of product-specific impurities. However, the combination of the functionalities makes industrial process development significantly more complex, in particular the identification of the often small elution window that delivers the desired selectivity. Mechanistic modeling has proven that even difficult separation problems can be solved in a computer-optimized manner once the process dynamics have been modeled. The adsorption models described in the literature are also very complex, which makes model calibration difficult. In this work, we approach this problem with a newly constructed model that describes the adsorber saturation with the help of the surface coverage function of the colloidal particle adsorption model for ion-exchange chromatography. In a case study, a model for a pH-controlled antibody polishing step was created from six experiments. The behavior of fragments, aggregates, and host cell proteins was described with the help of offline analysis. After in silico optimization, a validation experiment confirmed an improved process performance in comparison to the historical process set point. In addition to these good results, the work also shows that the high dynamics of mixed-mode chromatography can produce unexpected results if process parameters deviate too far from tried and tested conditions.

The continuing evolution of barcode applications: Functional toxicology to cell lineage.

DNA barcoding is a method to identify biological entities, including individual cells, tissues, organs, or species, by unique DNA sequences. With the advent of next generation sequencing (NGS), there has been an exponential increase in data acquisition pertaining to medical diagnosis, genetics, toxicology, ecology, cancer, and developmental biology. While barcoding first gained wide access in identifying species, signature tagged mutagenesis has been useful in elucidating gene function, particularly in microbes. With the advent of CRISPR/CAS9, methodology to profile eukaryotic genes has made a broad impact in toxicology and cancer biology. Designed homing guide RNAs (hgRNAs) that self-target DNA sequences facilitate cell lineage barcoding by introducing stochastic mutations within cell identifiers. While each of these applications has their limitations, the potential of sequence barcoding has yet to be realized. This review will focus on signature-tagged mutagenesis and briefly discuss the history of barcoding, experimental problems, novel detection methods, and future directions.

CYP1B1 converts procarcinogens into genotoxins in Saccharomyces cerevisiae.

CYP1B1 activates many chemical carcinogens into potent genotoxins, and allelic variants are risk factors in lung, breast, and prostate cancer. However, few eukaryotic genetic instability endpoints have been directly measured for CYP1B1-activated metabolites. In this study, we expressed human CYP1B1 in yeast strains that measure DNA damage-associated toxicity and frequencies of chromosomal translocations. DNA damage-associated toxicity was measured in a rad4 rad51 strain, defective in both DNA excision and recombinational repair. Frequencies of chromosomal translocations were measured in diploid yeast strains containing two his3 fragments. These strains were exposed to benzo[a]pyrene-7,8-dihydrodiol (BaP-DHD), aflatoxin B1 (AFB1), and the heterocyclic aromatic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). We observed that AFB1, BaP-DHD, IQ, and MeIQx conferred toxicity in the DNA repair mutant expressing CYP1B1. Translocation frequencies increased eight-fold and three-fold after exposure to 50 µM AFB1 and 33 µM BaP-DHD respectively. A DNA damage response was observed after AFB1 exposure, as measured by the induction of the small subunit of ribonucleotide reductase, Rnr3. While CYP1B1-mediated activation of BaP-DHD and heterocyclic aromatic amines was expected, activation of AFB1 to become a potent recombinagen was not expected. These studies demonstrate that chromosomal rearrangement is a useful genotoxic endpoint for CYP1B1-mediated carcinogen activation.

Effects of outdoor weathering and laundering on the detection and classification of fluorinated oil-and-water-repellent fabric coatings.

Fluorinated polymer coatings are used to impart durable oil-and-water-repellent properties on fabrics, potentially offering a persistent fiber characteristic for forensic fiber comparisons. To evaluate the persistence of these coatings, we investigate effects of outdoor weathering and laundering on detection and classification of the fluorinated oil-and-water-repellent coatings on 9 garments and 2 spray-coated fabric samples. Single fibers from the samples are pyrolyzed and subjected to gas chromatography coupled to a fluorine-selective detector. The positive detection of coatings is indicated by a signal-to-noise ratio (S/N) >50 for the tallest peak in the pyrograms. Moreover, a multinomial logistic regression model trained using fibers prior to weathering and laundering is utilized to determine the class of the weathered and laundered fibers, providing a metric to evaluate the effect of these processes on fiber classification. Notably, fluorinated coatings are detected on all of the fibers exposed to outdoor elements in Arlington, VA, up to 12 weeks from August to October 2020, while a detection rate of 95.5% is achieved for samples laundered up to 10 wash cycles. The detection rate prior to weathering and laundering was 98%, indicating negligible effect of these processes on detection of coatings. The classification accuracy is determined to be 99% and 100% for weathered and laundered samples, respectively, illustrating that these processes do not significantly affect the major pyrolysis products of the coatings responsible for classification. These results highlight the persistence of the fluorinated oil-and-water-repellent fabric coatings and their potential for forensic fiber discrimination at single-fiber level.

Detection and diversity of fluorinated oil- and water-repellent coatings on apparel fibers.

Fluorinated coatings, often used for oil and water repellency and stain resistance in fabrics, are potentially persistent forensic fiber markers. However, they have received limited attention because of challenges in their detection caused by the small size of a single fiber and thin nature of stain-resistant coatings. Here, we utilize a sensitive fluorine-selective analytical technique to detect and evaluate diversity of fluorinated coatings in apparel. Twelve clothing items marketed as stain-resistant were tested with nine showing oil- and water-repellent properties. Fluorinated pyrolysis products of single fibers from all of the nine items were detected by gas chromatography coupled to plasma-assisted reaction chemical ionization mass spectrometry (GC-PARCI-MS), indicating the prevalence of fluoropolymer coatings in stain-resistant clothing articles. Furthermore, three major classes of fluorinated coatings were identified via principal component analysis of pyrogram patterns. The classes were coating-specific and did not correlate with fiber core and color, highlighting a robust detection methodology. To evaluate the effect of fiber lifting in crime scenes, fibers from the 9 clothing items were used to develop a multinomial logistic regression model based on pyrogram principal components. The model was then tested using fibers subjected to contact with Post-it® notes. The test set fibers were sampled from the clothing items of the training set and from three additional garments of differing color but the same brands as the training set. The coating classes were predicted with 98.4% accuracy, confirming robust classification of fiber coatings using py-GC-PARCI-MS regardless of fiber color, core, and fiber lifting.

Functional Anatomy of the Trimer Apex Reveals Key Hydrophobic Constraints That Maintain the HIV-1 Envelope Spike in a Closed State.

The human immunodeficiency virus type 1 (HIV-1) envelope trimer maintains a closed, metastable configuration to protect vulnerable epitopes from neutralizing antibodies. Here, we identify key hydrophobic constraints at the trimer apex that function as global stabilizers of the HIV-1 envelope spike configuration. Mutation of individual residues within four hydrophobic clusters that fasten together the V1V2, V3, and C4 domains at the apex of gp120 dramatically increases HIV-1 sensitivity to weak and restricted neutralizing antibodies targeting epitopes that are largely concealed in the prefusion Env spike, consistent with the adoption of a partially open trimer configuration. Conversely, the same mutations decrease the sensitivity to broad and potent neutralizing antibodies that preferentially recognize the closed trimer. Sera from chronically HIV-infected patients neutralize open mutants with enhanced potency, compared to the wild-type virus, suggesting that a large fraction of host-generated antibodies target concealed epitopes. The identification of structural constraints that maintain the HIV-1 envelope in an antibody-protected state may inform the design of a protective vaccine.IMPORTANCE Elucidating the structure and function of the HIV-1 envelope proteins is critical for the design of an effective vaccine. Despite the availability of many high-resolution structures, key functional correlates in the envelope trimer remain undefined. We utilized a combination of structural analysis, in silico energy calculation, mutagenesis, and neutralization profiling to dissect the functional anatomy of the trimer apex, which acts as a global regulator of the HIV-1 spike conformation. We identify four hydrophobic clusters that stabilize the spike in a tightly closed configuration and, thereby, play a critical role in protecting it from the reach of neutralizing antibodies.

Cell types and neuronal circuitry underlying female aggression in Drosophila.

Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism's internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. Here, we identify a population of sexually dimorphic aIPg neurons in the adult Drosophila melanogaster central brain whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated aIPg and pC1d neurons as core nodes regulating female aggression. Connectomic analysis demonstrated that aIPg neurons and pC1d are interconnected and suggest that aIPg neurons may exert part of their effect by gating the flow of visual information to descending neurons. Our work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation.

Elemental Fluorine Detection by Dielectric Barrier Discharge Coupled to Nanoelectrospray Ionization Mass Spectrometry for Nontargeted Analysis of Fluorinated Compounds.

The growing use of fluorochemicals has elevated the need for nontargeted detection of unknown fluorinated compounds and transformation products. Elemental mass spectrometry (MS) coupled to chromatography offers a facile approach for such analyses by using fluorine as an elemental tag. However, efficient ionization of fluorine has been an ongoing challenge. Here, we demonstrate a novel atmospheric-pressure elemental ionization method where fluorinated compounds separated by gas chromatography (GC) are converted to Na2F+ for nontargeted detection. The compounds are first introduced into a helium dielectric barrier discharge (DBD) for breakdown. The plasma products are subsequently ionized by interaction with a nanoelectrospray ionization (nano-ESI) plume of sodium-containing aqueous electrolytes. Our studies point to HF as the main plasma product contributing to Na2F+ formation. Moreover, the results reveal that Na2F+ is largely formed by the ion-neutral reaction between HF and Na2A(NaA)n+, gas-phase reagent ions produced by nano-ESI where A represents the anion of the electrolyte. Near-uniform fluorine response factors are obtained for a wide range of compounds, highlighting good efficiency of HF formation by DBD regardless of the chemical structure of the compounds. Detection limits of 3.5-19.4 pg of fluorine on-column are obtained using the reported GC-DBD-nano-ESI-MS. As an example of nontargeted screening, extractions from oil-and-water-repellent fabrics are analyzed via monitoring Na2F+, resulting in detection of a fluorinated compound on a clothing item. Notably, facile switching of the ion source to atmospheric-pressure chemical ionization with the exact same chromatographic method allows identification of the detected compound at the flagged retention time.

Site-specific conjugation of native antibody.

Traditionally, non-specific chemical conjugation, such as acylation of amines on lysine or alkylation of thiols on cysteines, are widely used; however, they have several shortcomings. First, the lack of site-specificity results in heterogeneous products and irreproducible processes. Second, potential modifications near the complementarity determining region (CDR) may reduce binding affinity and specificity. Conversely, site-specific methods produce well-defined and more homogenous antibody conjugates, ensuring developability and clinical applications. Moreover, several recent side-by-side comparisons of site-specific and stochastic methods have demonstrated that site-specific approaches are more likely to achieve their desired properties and functions, such as increased plasma stability, less variability in dose-dependent studies (particularly at low concentrations), enhanced binding efficiency, as well as increased tumor uptake. Herein we review several standard and practical site-specific bioconjugation methods for native antibodies, i.e., those without recombinant engineering. First, chemo-enzymatic techniques, namely transglutaminase (TGase)-mediated transamidation of a conserved glutamine residue and glycan remodeling of a conserved asparagine N-glycan (GlyCLICK), both in the Fc region. Second, chemical approaches such as selective reduction of disulfides (ThioBridge) and N-terminal amine modifications. Furthermore, we list site-specific antibody-drug conjugates (ADCs) in clinical trials along with the future perspectives of these site-specific methods.

Functional and anatomical specificity in a higher olfactory centre.

Most sensory systems are organized into parallel neuronal pathways that process distinct aspects of incoming stimuli. In the insect olfactory system, second order projection neurons target both the mushroom body, required for learning, and the lateral horn (LH), proposed to mediate innate olfactory behavior. Mushroom body neurons form a sparse olfactory population code, which is not stereotyped across animals. In contrast, odor coding in the LH remains poorly understood. We combine genetic driver lines, anatomical and functional criteria to show that the Drosophila LH has ~1400 neurons and >165 cell types. Genetically labeled LHNs have stereotyped odor responses across animals and on average respond to three times more odors than single projection neurons. LHNs are better odor categorizers than projection neurons, likely due to stereotyped pooling of related inputs. Our results reveal some of the principles by which a higher processing area can extract innate behavioral significance from sensory stimuli.

Neurogenetic dissection of the Drosophila lateral horn reveals major outputs, diverse behavioural functions, and interactions with the mushroom body.

Animals exhibit innate behaviours to a variety of sensory stimuli including olfactory cues. In Drosophila, one higher olfactory centre, the lateral horn (LH), is implicated in innate behaviour. However, our structural and functional understanding of the LH is scant, in large part due to a lack of sparse neurogenetic tools for this region. We generate a collection of split-GAL4 driver lines providing genetic access to 82 LH cell types. We use these to create an anatomical and neurotransmitter map of the LH and link this to EM connectomics data. We find ~30% of LH projections converge with outputs from the mushroom body, site of olfactory learning and memory. Using optogenetic activation, we identify LH cell types that drive changes in valence behavior or specific locomotor programs. In summary, we have generated a resource for manipulating and mapping LH neurons, providing new insights into the circuit basis of innate and learned olfactory behavior.

Peripapillary and macular morpho-vascular changes in patients with genetic or clinical diagnosis of autosomal dominant optic atrophy: a case-control study.

PURPOSE: To evaluate the macular and peripapillary morpho-vascular changes in ADOA, using optical coherence tomography (OCT) and OCT angiography (OCTA). METHODS: Prospectively defined, cross-sectional case-control study. Consecutive patients with a genetic or clinical diagnosis of ADOA along with age- and sex-matched controls were included. The radial peripapillary capillary (RPC) density and vessel density (VD) in the parafoveal superficial and deep capillary plexuses (SCP and DCP, respectively) were evaluated with OCTA. The ganglion cell complex (GCC) and retinal nerve fiber layer (RNFL) thickness were determined using structural OCT. We applied a previously validated customized macro (Fiji, SciJava Consortium) to compute RPC density. The remaining parameters were calculated by the built-in software. Non-parametric methods were used for data analysis. The target α level was 0.05, which was adjusted through Bonferroni's correction when multiple outcomes were tested. RESULTS: Fifty-eight eyes (n = 29 control; n = 29 ADOA) from 30 subjects (mean age 42.43 ± 15.30 years; 37.93% male) were included. Parafoveal SCP VD, GCC thickness, RPC VD in the temporal quadrant, as well as RNFL thickness in the nasal and temporal quadrants were decreased in ADOA eyes (all p < 0.001). When only patients with genetically confirmed diagnosis were included, capillary dropout in the circumpapillary superior and inferior quadrants also became evident (both p < 0.001). The GCC/parafoveal SCP ratio was increased in ADOA, relatively to matched controls. In contrast, none of the circumpapillary morpho-vascular ratios was significantly different in ADOA eyes. CONCLUSIONS: The microvascular and structural changes found in ADOA suggest that both the macular and peripapillary regions are involved, although the threshold for damage of the structural and vascular components may be different for each region. Larger series with longitudinal follow-up may validate OCTA biomarkers helpful for disease monitoring.

Classifying single fibers based on fluorinated surface treatments.

Fibers are an important form of forensic evidence, but their evidential value can be severely limited when the identified characteristics of the fibers are common, such as blue cotton. Detecting chemical fiber treatments offers an avenue to further classify fibers and to improve their evidential value. In this report, we investigate the potential of fluoropolymer fiber coatings, used to impart oil and water-repellent properties in fabrics, for differentiating between fibers. The thin nature of these fiber surface modifications creates an analytical challenge for their detection on a single fiber, a typical sample size for forensic evidence. Specifically, pyrolysis-gas chromatography-mass spectrometry (py-GC-MS) has shown promising selectivity but the sensitivity of the method is not adequate for single-fiber analysis of fluorinated coatings. To overcome this challenge, we utilize a newly developed elemental ionization source, plasma-assisted reaction chemical ionization (PARCI). The high sensitivity of py-GC-PARCI-MS for elemental fluorine analysis offers selective and sensitive detection of fluorinated pyrolysates among the non-fluorinated pyrolysates of the fiber core. As a result, fluoropolymer coatings are detected from 10-mm single-fiber samples. The technique is applied for classification of 22 fiber types, resulting in 4 distinct groups via hierarchical cluster analysis based on similarity of fluorine pyrograms. These results present the first study to classify fibers based on fluorinated coatings, and highlight the potential of py-GC-PARCI-MS for forensic analyses. Graphical Abstract ᅟ.

CNS synapses are stabilized trans-synaptically by laminins and laminin-interacting proteins.

The retina expresses several laminins in the outer plexiform layer (OPL), where they may provide an extracellular scaffold for synapse stabilization. Mice with a targeted deletion of the laminin ß2 gene (Lamb2) exhibit retinal disruptions: photoreceptor synapses in the OPL are disorganized and the retinal physiological response is attenuated. We hypothesize that laminins are required for proper trans-synaptic alignment. To test this, we compared the distribution, expression, association and modification of several pre- and post-synaptic elements in wild-type and Lamb2-null retinae. A potential laminin receptor, integrin α3, is at the presynaptic side of the wild-type OPL. Another potential laminin receptor, dystroglycan, is at the post-synaptic side of the wild-type OPL. Integrin α3 and dystroglycan can be co-immunoprecipitated with the laminin ß2 chain, demonstrating that they may bind laminins. In the absence of the laminin ß2 chain, the expression of many pre-synaptic components (bassoon, kinesin, among others) is relatively undisturbed although their spatial organization and anchoring to the membrane is disrupted. In contrast, in the Lamb2-null, ß-dystroglycan (ß-DG) expression is altered, co-localization of ß-DG with dystrophin and the glutamate receptor mGluR6 is disrupted, and the post-synaptic bipolar cell components mGluR6 and GPR179 become dissociated, suggesting that laminins mediate scaffolding of post-synaptic components. In addition, although pikachurin remains associated with ß-DG, pikachurin is no longer closely associated with mGluR6 or α-DG in the Lamb2-null. These data suggest that laminins act as links among pre- and post-synaptic laminin receptors and α-DG and pikachurin in the synaptic space to maintain proper trans-synaptic alignment.

Comparison of seating, powered characteristics and functions and costs of electrically powered wheelchairs in a general population of users.

PURPOSE: To profile and compare the seating and powered characteristics and functions of electrically powered wheelchairs (EPWs) in a general user population including equipment costs. METHOD: Case notes of adult EPW users of a regional NHS service were reviewed retrospectively. Seating equipment complexity and type were categorized using the Edinburgh classification. Powered characteristics and functions, including control device type, were recorded. RESULTS: 482 cases were included; 53.9% female; mean duration EPW use 8.1 years (SD 7.4); rear wheel drive 88.0%; hand joystick 94.8%. Seating complexity: low 73.2%, medium 18.0%, high 8.7%. Most prevalent diagnoses: multiple sclerosis (MS) 25.3%, cerebral palsy (CP) 18.7%, muscular dystrophy (8.5%). Compared to CP users, MS users were significantly older at first use, less experienced, more likely to have mid-wheel drive and less complex seating. Additional costs for muscular dystrophy and spinal cord injury users were 3-4 times stroke users. CONCLUSIONS: This is the first large study of a general EPW user population using a seating classification. Significant differences were found between diagnostic groups; nevertheless, there was also high diversity within each group. The differences in provision and the equipment costs across diagnostic groups can be used to improve service planning. Implications for Rehabilitation At a service planning level, knowledge of a population's diagnostic group and age distribution can be used to inform decisions about the number of required EPWs and equipment costs. At a user level, purchasing decisions about powered characteristics and functions of EPWs and specialised seating equipment need to be taken on a case by case basis because of the diversity of users' needs within diagnostic groups. The additional equipment costs for SCI and MD users are several times those of stroke users and add between 60 and 70% of the cost of basic provision.

PERIPAPILLARY NEUROVASCULAR COUPLING IN THE EARLY STAGES OF DIABETIC RETINOPATHY.

PURPOSE: To study radial peripapillary capillary (RPC) density in the early stages of diabetic retinopathy (DR), using optical coherence tomography angiography. METHODS: A cross-sectional evaluation of RPCs was performed using optical coherence tomography angiography (Avanti RTVue-XR 100, Optovue Inc, Fremont, CA). Annular RPC density was the primary outcome. Global density and retinal nerve fiber layer thickness were secondary outcomes. Diabetic eyes were divided into three groups: no DR, mild nonproliferative DR (mild NPDR), and moderate NPDR. Multilevel mixed-effects univariate and multivariate linear regression models were used. RESULTS: We included 155 eyes (n = 42 control; n = 27 no DR; n = 28 mild NPDR; and n = 58 moderate NPDR) from 86 subjects (mean [SD] age 63.39 [10.70] years; 46.45% male). When compared with controls, a significant decrease in annular RPC density was found in all groups of diabetic eyes on multivariate analysis (no DR: ß = -2.95, P < 0.001; mild NPDR: ß = -1.76, P = 0.017; and moderate NPDR: ß = -2.82, P < 0.001). We also detected a significant decrease in retinal nerve fiber layer thickness in diabetic eyes (even in the no DR group). Furthermore, in diabetic eyes, annular RPC density and retinal nerve fiber layer thickness correlated significantly (R = 0.4874, P < 0.001). CONCLUSION: Peripapillary neurovascular changes occur early in the course of DR. Their significance in the progression of DR warrants further research.

Structural Constraints at the Trimer Apex Stabilize the HIV-1 Envelope in a Closed, Antibody-Protected Conformation.

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) trimer evades antibody recognition by adopting a closed prefusion conformation. Here, we show that two conserved tyrosines (Y173, Y177) within the second variable (V2) loop of the gp120 Env glycoprotein are key regulators of the closed, antibody-protected state of the trimer by establishing intramolecular interaction with the base of the third variable (V3) loop. Mutation of Y177 and/or Y173 to phenylalanine or alanine dramatically altered the susceptibility of diverse HIV-1 strains to neutralization, increasing sensitivity to weakly and nonneutralizing antibodies directed against diverse Env regions, consistent with the adoption of an open trimer configuration. Conversely, potent broadly neutralizing antibodies (bNAbs) against different supersites of HIV-1 vulnerability exhibited reduced potency against V2 loop tyrosine mutants, consistent with their preferential targeting of the closed trimer. Mutation of V3 loop residues predicted to interact with the V2 loop tyrosines yielded a similar neutralization phenotype. Sera from chronically HIV-1-infected patients contained very high titers of antibodies capable of neutralizing V2 loop tyrosine mutants but not wild-type viruses, indicating that the bulk of antibodies produced in infected hosts are unable to penetrate the protective shield of the closed trimer. These results identify the tyrosine-mediated V2-V3 loop complex at the trimer apex as a key structural constraint that facilitates HIV-1 evasion from the bulk of host antibodies.IMPORTANCE The extraordinary ability of human immunodeficiency virus type 1 (HIV-1) to evade host immunity represents a major obstacle to the development of a protective vaccine. Thus, elucidating the mechanisms whereby HIV-1 protects its external envelope (Env), which is the sole target of virus-neutralizing antibodies, is an essential step toward vaccine design. We identified a key structural element that maintains the HIV-1 Env trimer in a closed, antibody-resistant conformation. A major role is played by two conserved tyrosines at the apex of the Env spike, whose mutation causes a global opening of the trimer structure, exposing multiple concealed targets for neutralizing antibodies. We also found that HIV-infected individuals produce very large amounts of antibodies that neutralize the open Env form; however, the bulk of these antibodies are unable to penetrate the tight defensive shield of the native virus. This work may help to devise new strategies to overcome the viral defensive mechanisms and facilitate the development of an effective HIV-1 vaccine.

Diabetes Mellitus-Not Just Type 1 or Type 2 Anymore.

INTRODUCTION: Diabetes mellitus traditionally has been categorized as type 1 (insulin deficiency due to autoimmune destruction of islet cells) or type 2 (insulin resistance with the development of relative insulin deficiency). However, other pathophysiologic etiologies for diabetes must be considered in the evaluation of patients with new-onset diabetes. CASE PRESENTATION: We report the case of a 50-year-old man with a diagnosis of type 2 diabetes mellitus who-despite appropriate pharmacotherapy-developed worsening hyperglycemia. Further investigation revealed the presence of metastatic pancreatic cancer. DISCUSSION: Although an association between pancreatic cancer and diabetes has been noted widely in the gastroenterology, oncology, and endocrine literature, a paucity of primary care literature on the topic exists. Features of predominant insulin deficiency and new onset of diabetes in a patient without family history of type 2 diabetes should raise suspicion for undetected/early-stage pancreatic cancer. CONCLUSIONS: This case highlights the importance of considering all possible pathophysiologic etiologies when a patient has a new diagnosis of diabetes. Clinicians should consider the possibility of pancreatic cancer in patients with new-onset diabetes mellitus, especially when features not characteristic of type 2 diabetes are present. Understanding the relationship between diabetes and pancreatic cancer has the potential to improve early detection of pancreatic cancer and can provide an opportunity for early treatment and improved survival.