The rockefeller university

Pre-term birth is a major health concern that occurs in approximately 10% of births worldwide. Despite high incidence rate, long-term consequences of pre-term birth remain unclear. Recent evidence suggests that elevated oxidative stress observed in pre-term born infants could persist into adulthood. Given that oxidative stress is known to play an important role in response to physical activity and hypoxia, we investigated whether oxidative stress responses to acute exercise in normoxia and hypoxia may be differently modulated in pre-term vs. full-term born adults. Twenty-two pre-term born and fifteen age-matched full-term born controls performed maximal incremental cycling tests in both normoxia (FiO2: 0.21) and normobaric hypoxia (FiO2: 0.13; simulated altitude of 3800 m) in blinded and randomized manner. Plasma levels of oxidative stress (advanced oxidation protein products [AOPP] and malondialdehyde), antioxidant (ferric reducing antioxidant power, glutathione peroxidase, catalase [CAT] and superoxide dismutase [SOD]) and nitrosative stress markers (nitrotyrosine, nitrite and total nitrite and nitrate [NOx]) were measured before and immediately after each test. AOPP (+24%, P < 0.001), CAT (+38%, P < 0.001) and SOD (+12%, P=0.018) and NOx (+17%, P=0.024) significantly increased in response to exercise independently of condition and birth status. No difference in response to acute exercise in normoxia was noted between pre-term and full-term born adults in any of measured markers. Hypoxic exposure during exercise resulted in significant increase in AOPP (+45%, P=0.008), CAT (+55%, P=0.019) and a trend for an increase in nitrite/nitrate content (+35%, P=0.107) only in full-term and not pre-term born individuals. These results suggest that prematurely born adult individuals exhibit higher resistance to oxidative stress response to exercise in hypoxia.

Most monogenic disorders have a primary clinical presentation. Inherited ISG15 deficiency, however, has manifested with two distinct presentations to date: susceptibility to mycobacterial disease and intracranial calcifications from hypomorphic interferon-II (IFN-II) production and excessive IFN-I response, respectively. Accordingly, these patients were managed for their infectious and neurologic complications. Herein, we describe five new patients with six novel ISG15 mutations presenting with skin lesions who were managed for dermatologic disease. Cellularly, we denote striking specificity to the IFN-I response, which was previously assumed to be universal. In peripheral blood, myeloid cells display the most robust IFN-I signatures. In the affected skin, IFN-I signaling is observed in the keratinocytes of the epidermis, endothelia, and the monocytes and macrophages of the dermis. These findings define the specific cells causing circulating and dermatologic inflammation and expand the clinical spectrum of ISG15 deficiency to dermatologic presentations as a third phenotype co-dominant to the infectious and neurologic manifestations.

Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P-2) is critical for synaptic vesicle docking and fusion and generation of the second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. PI-4,5-P(2)can be generated by two families of kinases: type 1 phosphatidylinositol-4-phosphate 5-kinases, encoded by PIP5K1A, PIP5K1B and PIP5K1C, and type 2 phosphatidylinositol-5-phosphate 4-kinases, encoded by PIP4K2A, PIP4K2B, and PIP4K2C. While the roles of the type 1 enzymes in brain function have been extensively studied, the roles of the type 2 enzymes are poorly understood. Using selective antibodies validated by genetic deletion of pip4k2a or pip4k2b in mouse brain, we characterized the location of the enzymes, PI5P4K alpha and PI5P4K beta, encoded by these genes. In mice, we demonstrate that PI5P4K alpha is expressed in adulthood, whereas PI5P4K beta is expressed early in development. PI5P4K alpha localizes to white matter tracts, especially the corpus callosum, and at a low level in neurons, while PI5P4K beta is expressed in neuronal populations, especially hippocampus and cortex. Dual labeling studies demonstrate that PI5P4K alpha co-localizes with the oligodendrocyte marker, Olig2, whereas PI5P4K beta co-localizes with the neuronal marker, NeuN. Ultrastructural analysis demonstrates that both kinases are contained in axon terminals and dendritic spines adjacent to the synaptic membrane, which support a potential role in synaptic transmission. Immunoperoxidase analysis of macaque and human brain tissue demonstrate a conserved pattern for PI5P4K alpha and PI5P4K beta. These results highlight the diverse cell-autonomous expression of PI5P4K alpha and PI5P4K beta and support further exploration into their role in synaptic function in the brain.

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. Here we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs.

Author summary Due to its extreme reductive evolution Mycobacterium leprae is a rare example of an essentially clonal bacterial pathogen that affects humans. However, exposed individuals display a wide diversity of symptoms reflecting the interactions between the host immune response and the bacterium. There is now accumulating evidence, in particular from genome-wide association study (GWAS), that common genetic variants play a role explaining this variability. Previous GWAS were based on case control design and almost all of them were conducted in the Chinese population. We conducted the first family-based GWAS of leprosy in the Vietnamese population and identified several genome-wide significant association signals within the HLA region. By performing a multivariate analysis in an independent case-control sample of the same ethnic origin we were able to decipher the HLA association signal and to reduce it to three independent SNPs, two in the class I and one in the class II region. We also validated two previously identified risk factors for leprosy, a missense variant in the LACC1 gene, and an intergenic variant located close to the IL12B gene. These results shed new light on the genetic control of leprosy and, in particular, on the influence of HLA SNPs in a large Vietnamese sample. Leprosy is a chronic infectious disease of the skin and peripheral nerves with a strong genetic predisposition. Recent genome-wide approaches have identified numerous common variants associated with leprosy, almost all in the Chinese population. We conducted the first family-based genome-wide association study of leprosy in 622 affected offspring from Vietnam, followed by replication in an independent sample of 1181 leprosy cases and 668 controls of the same ethnic origin. The most significant results were observed within the HLA region, in which six SNPs displayed genome-wide significant associations, all of which were replicated in the independent case/control sample. We investigated the signal in the HLA region in more detail, by conducting a multivariate analysis on the case/control sample of 319 GWAS-suggestive HLA hits for which evidence for replication was obtained. We identified three independently associated SNPs, two located in the HLA class I region (rs1265048: OR = 0.69 [0.58-0.80], combined p-value = 5.53x10(-11); and rs114598080: OR = 1.47 [1.46-1.48], combined p-value = 8.77x10(-13)), and one located in the HLA class II region (rs3187964 (OR = 1.67 [1.55-1.80], combined p-value = 8.35x10(-16)). We also validated two previously identified risk factors for leprosy: the missense variant rs3764147 in the LACC1 gene (OR = 1.52 [1.41-1.63], combined p-value = 5.06x10(-14)), and the intergenic variant rs6871626 located close to the IL12B gene (OR = 0.73 [0.61-0.84], combined p-value = 6.44x10(-8)). These results shed new light on the genetic control of leprosy, by dissecting the influence of HLA SNPs, and validating the independent role of two additional variants in a large Vietnamese sample.

Therapeutic interventions in colorectal cancer are dependent on immune responses to dying epithelial cells that are modulated by specific members of the gut microbiota.

The application of machine learning to longitudinal gene-expression profiles has demonstrated potential to decrease the assessment gap, between biochemical determination and clinical manifestation, of a patient's response to treatment. Although psoriasis is a proven testing ground for treatment-response prediction using transcriptomic data from clinically accessible skin biopsies, these biopsies are expensive, invasive, and challenging to obtain from certain body areas. Response prediction from blood biochemical measurements could be a cheaper, less invasive predictive platform. Longitudinal profiles for 92 inflammatory and 65 cardiovascular disease proteins were measured from the blood of psoriasis patients at baseline, and 4-weeks, following tofacitinib (janus kinase-signal transducer and activator of transcription-inhibitor) or etanercept (tumor necrosis factor-inhibitor) treatment, and predictive models were developed by applying machine-learning techniques such as bagging and ensembles. This data driven approach developed predictive models able to accurately predict the 12-week clinical endpoint for psoriasis following tofacitinib (area under the receiver operating characteristic curve [auROC] = 78%), or etanercept (auROC = 71%) treatment in a validation dataset, revealing a robust predictive protein signature including well-established psoriasis markers such as IL-17A and IL-17C, highlighting potential for biologically meaningful and clinically useful response predictions using blood protein data. Although most blood classifiers were outperformed by simple models trained using Psoriasis Area Severity Index scores, performance might be enhanced in future studies by measuring a wider variety of proteins.

The discovery of specific microbiota metabolite mechanisms has begun to motivate new therapeutic approaches. Inspired by our mechanistic studies of microbiota-derived short chain fatty acid (SCFA) acylation of bacterial virulence factors, here we explored covalent protein acylation therapeutics as potential anti-infectives. For these studies, we focused on acetyl-salicylic acid, aspirin, and discovered that SCFA analogues such as butyryl-salicylic acid showed significantly improved anti-infective activity against Salmonella Typhimurium. Structure-activity studies showed that the ester functionality of butyryl-salicylic acid was crucial and associated with the acylation of key bacterial virulence factors and metabolic enzymes, which are important for Salmonella infection of host cells and bacterial growth. Beyond the Gram-negative bacterial pathogens, butyryl-salicylic acid also showed better antibacterial activity compared to aspirin against Clostridioides difficile, a clinically challenging Gram-positive bacterial pathogen. Notably, coadministration of butyryl-salicylic acid, but not aspirin, effectively attenuated Salmonella pathogenesis in vivo. This study highlights how the analysis of microbiota metabolite mechanisms may inspire the repurposing and development of new anti-infective agents.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named 'Worm-to-CT', allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

Interferon-induced transmembrane protein 3 (IFITM3) is a key interferon effector that broadly prevents infection by diverse viruses. However, the cellular factors that control IFITM3 homeostasis and antiviral activity have not been fully elucidated. Using site-specific photo-crosslinking and quantitative proteomic analysis, here we present the identification and functional characterization of VCP/p97 AAA-ATPase as a primary interaction partner of IFITM3. We show that IFITM3 ubiquitination at lysine 24 is crucial for VCP binding, trafficking, turnover, and engagement with incoming virus particles. Consistently, pharmacological inhibition of VCP/p97 ATPase activity leads to defective IFITM3 lysosomal sorting, turnover, and co-trafficking with virus particles. Our results showcase the utility of site-specific protein photo-crosslinking in mammalian cells and reveal VCP/p97 as a key cellular factor involved in IFITM3 trafficking and homeostasis.