Nonperturbative Fluorogenic Labeling of Immunophilins Enables the Wash-free Detection of Immunosuppressants.

Immunosuppressants are clinically approved drugs to treat the potential rejection of transplanted organs and require frequent monitoring due to their narrow therapeutic window. Immunophilins are small proteins that bind immunosuppressants with high affinity, yet there are no examples of fluorogenic immunophilins and their potential application as optical biosensors for immunosuppressive drugs in clinical biosamples. In the present work, we designed novel diazonium BODIPY salts for the site-specific labeling of tyrosine residues in peptides via solid-phase synthesis as well as for late-stage functionalization of whole recombinant proteins. After the optimization of a straightforward one-step labeling procedure for immunophilins PPIA and FKBP12, we demonstrated the application of a fluorogenic analogue of FKBP12 for the selective detection of the immunosuppressant drug tacrolimus, including experiments in urine samples from patients with functioning renal transplants. This chemical methodology opens new avenues to rationally design wash-free immunophilin-based biosensors for rapid therapeutic drug monitoring.

Inserting "OFF-to-ON" BODIPY Tags into Cytokines: A Fluorogenic Interleukin IL-33 for Real-Time Imaging of Immune Cells.

The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first nativelike fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results. Importantly, the BODIPY-labeled IL-33 derivatives-unlike IL-33-GFP constructs-exhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells.

Hepatitis B in Trans-Himalayan Tribal Population: High Prevalence, High Pediatric Burden, Virologic Patterns, and Implications for Public Health Interventions.

Background/Aims: The prevalence of hepatitis B is higher in tribal populations, compared to non-tribal populations in India. Therefore, this study aimed to investigate the risk factors, virological and biochemical profile of patients with hepatitis B in a tribal population. Methods: This study analyzed data collected from a community-based project conducted in Spiti, Himachal Pradesh, from July 2015 to 2017. The study included adults and children inhabiting 40 cluster villages out of 82 villages in the subdivision. The blood samples were collected for liver panel, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), Anti-HBe antibody (anti-HBe Ab) and Hepatitis B virus DNA (HBV-DNA). Results: HBsAg was positive in 23.08% of the population (968/4201), with a prevalence of 13.51% in children under 5 years of age. HBeAg positivity was seen in 22.4% of the participants, while anti-HBe Ab positivity was seen in 59.03% of the participants. HBeAg positive infection, HBeAg positive hepatitis, HBeAg negative hepatitis and HBeAg negative infection were seen in 18.06%, 1.98%, 6.17% and 74.01% of the participants, respectively. HBeAg positivity was highest in 2nd decade (40.83% vs 22% overall). Patients with HBeAg positivity exhibited higher levels of HBV DNA [1960 (IQR: 0-108) IU/ml vs 97.2 (IQR: 0-2090) IU/ml, P < 0.001] and alanine transaminase (ALT) [22.5 (IQR: 16-33) U/L vs 19 (IQR: 14-26) U/L, P = 0.003] levels compared to HBeAg negative patients. Conclusion: This study shows a high prevalence of hepatitis B in tribal population, particularly among children under 5 years of age.

Engineering Homogeneous Photoactive Antibody Fragments.

Genetically encoded site-specifically incorporated noncanonical amino acids (ncAAs) have been used to modulate properties of several proteins. Here, we describe a procedure for engineering photoactive antibody fragments that bind to their target antigen only after irradiation with 365 nm light. The procedure starts with identification of tyrosine residues in antibody fragments that are important for antibody-antigen binding and thus targets for replacement with photocaged tyrosine (pcY). This is followed by cloning of plasmids and expression of pcY-containing antibody fragments in E. coli. Finally, we describe a cost-effective and biologically-relevant method for measuring the binding affinity of photoactive antibody fragments to antigens expressed on the surface of live cancer cells.

P2Y12 Inhibitors in Acute Coronary Syndromes: A Real-World, Community-Based Comparison of Ischemic and Bleeding Outcomes.

Background: Randomized trials have shown superiority of the novel P2Y12 inhibitors over clopidogrel in patients with acute coronary syndrome (ACS), but clinical benefit in the community remains controversial. Our objective was to compare the safety and efficacy of clopidogrel to ticagrelor and prasugrel in patients with ACS undergoing percutaneous coronary intervention (PCI) in a real-world population. Methods: We conducted a retrospective cohort study of patients with ACS who underwent PCI and were discharged with clopidogrel, ticagrelor, or prasugrel from 2012 to 2018 within Kaiser Permanente Northern California. We used Cox proportional hazard models with propensity-score matching to evaluate the association of the P2Y12 agent with the primary outcomes of all-cause mortality, myocardial infarction (MI), stroke, and bleeding events. Results: The study included 15,476 patients (93.1% on clopidogrel, 3.6% on ticagrelor and 3.2% on prasugrel). Compared to the clopidogrel group, ticagrelorand prasugrel patients were younger with less comorbidities. In multivariable models with propensity-score matching, we found a lower risk of all-cause mortality in the ticagrelor vs the clopidogrel group (HR (95% CI) 0.43 (0.20-0.92)), but no differences in the other endpoints, and no difference between prasugrel and clopidogrel among any endpoints. A larger proportion of patients on ticagrelor or prasugrel switched to an alternative P2Y12 agent vs. clopidogrel (p < 0.01), and a higher level of persistence was seen among patients on clopidogrel vs. ticagrelor (p = 0.03) or prasugrel (p < 0.01). Conclusion: Among patients with ACS who underwent PCI, we observed a lower risk of all-cause mortality in patients treated with ticagrelor vs clopidogrel, but no difference in other clinical endpoints nor any differences in endpoints between prasugrel vs. clopidogrel users. These results suggest that further study is needed to identify an optimal P2Y12 inhibitor in a real-world population.

Site-specific encoding of photoactivity and photoreactivity into antibody fragments.

Design of biomolecules that perform two or more distinct functions in response to light remains challenging. Here, we have introduced concurrent photoactivity and photoreactivity into an epidermal growth factor receptor (EGFR)-targeting antibody fragment, 7D12. This was achieved by site-specific incorporation of photocaged tyrosine (pcY) for photoactivity and p-benzoyl-ʟ-phenylalanine (Bpa) for photoreactivity into 7D12. We identified a position for installing Bpa in 7D12 that has minimal effect on 7D12-EGFR binding affinity in the absence of light. Upon exposure to 365-nm light, this Bpa-containing 7D12 mutant forms a covalent bond with EGFR in an antigen-specific manner. We then developed a method for site-specific incorporation of pcY and Bpa at two distinct sites in 7D12. Finally, we demonstrated that in the absence of light, this pcY- and Bpa-containing mutant of 7D12 does not bind to EGFR, but irradiation with 365-nm light activates (1) specific binding and (2) covalent bond formation with EGFR.

Cost of Surgical Care at Public Sector District Hospitals in India: Implications for Universal Health Coverage and Publicly Financed Health Insurance Schemes.

BACKGROUND: In low- and middle-income countries (LMICs), provisioning for surgical care is a public health priority. Ayushman Bharat Pradhan Mantri-Jan Aarogya Yojana (AB PM-JAY) is India's largest national insurance scheme providing free surgical and medical care. In this paper, we present the costs of surgical health benefit packages (HBPs) for secondary care in public district hospitals. METHODS: The costs were estimated using mixed (top-down and bottom-up) micro-costing methods. In phase II of the Costing of Health Services in India (CHSI) study, data were collected from a sample of 27 district hospitals from nine states of India. The district hospitals were selected using stratified random sampling based on the district's composite development score. We estimated unit costs for individual services-outpatient (OP) visit, per bed-day in inpatient (IP) and intensive care unit (ICU) stays, and surgical procedures. Together, this was used to estimate the cost of 250 AB PM-JAY HBPs. RESULTS: At the current level of utilization, the mean cost per OP consultation varied from US$4.10 to US$2.60 among different surgical specialities. The mean unit cost per IP bed-day ranged from US$13.40 to US$35.60. For the ICU, the mean unit cost per bed-day was US$74. Further, the unit cost of HBPs varied from US$564 for bone tumour excision to US$49 for lid tear repair. CONCLUSIONS: Data on the cost of delivering surgical care at the level of district hospitals is of critical value for evidence-based policymaking, price-setting for surgical care and planning to strengthen the availability of high quality and cost-effective surgical care in district hospitals.

Sensorineural hearing loss and ulcerative colitis in remission.

INTRODUCTION: Sensorineural hearing loss (SNHL) has been reported in association with inflammatory bowel disease (IBD). However, SNHL as an extraintestinal manifestation of IBD is frequently underreported. In the present study, we compared the prevalence and severity of SNHL among patients with IBD-ulcerative colitis (IBD-UC) in remission with controls to find out any association between SNHL and IBD-UC in remission compared to controls. METHODS: This single-center hospital-based prospective observational study included outdoor patients with IBD-UC in remission and healthy age- and sex-matched controls. Eligible patients and healthy participants were subjected to a battery of audiological tests (otoscopy, tympanometry and pure tone audiometry [PTN]) after thorough systemic and ear, nose and throat (ENT) examination. RESULTS: A total of 100 patients were enrolled in the study: 50 in IBD-UC in the remission group and 50 in the control group. None of the demographic variables (age, gender, residence and habits) were significantly different between the two groups. Otoscopy and tympanometry were normal in all patients and controls. The difference between the two groups in respect to frequency and severity of SNHL on PTA and in respect to unilateral and bilateral distribution of the hearing loss was not statistically significant. CONCLUSION: There is no statistically significant difference in frequency and severity of SNHL between patients with ulcerative colitis in remission and healthy age- and sex-matched controls.

Estimation of Baseline Widal Antibody Titers among Apparently Healthy Urban Population of District Jammu.

INTRODUCTION: Definitive diagnosis of Enteric fever is by blood culture for Salmonella enterica serotype Typhi, Paratyphi A, and Paratyphi B which takes long turnaround time and is costly, whereas Widal test is simple, rapid, and cost-effective test whose interpretation depends on the baseline Widal titers among healthy individuals in a defined population. OBJECTIVES: To determine the baseline Widal titers among apparently healthy urban population of district Jammu (J&K). MATERIALS AND METHODS: 302 individuals in the age group of 18-50 years were recruited. A pretested questionnaire was used to collect demographic and clinical details. The Widal testing was done using commercial Salmonella antigen kit. RESULTS: A total of 302 samples were screened by Widal test. 138 samples (45.69%) were reactive for TO antigen and 64 (21.19%) tested reactive for TH antigen, 3 (0.01%) samples showed agglutination for AH antigen and 3 (0.01%) were positive for BH antigen. Majority of seropositive samples were in dilutions of 1:40 for both TO and TH antigens. CONCLUSIONS: Hence, next higher dilutions showing positivity for both TO and TH antigens, i.e., ≥1:80 may be considered diagnostic for enteric fever in the urban population of Jammu district.

Duration of Dual Antiplatelet Therapy After Percutaneous Coronary Intervention for Chronic Total Occlusion.

The optimal duration of dual antiplatelet therapy (DAPT) after treatment of chronic total occlusions (CTO) with percutaneous coronary intervention (PCI) is unknown. We aimed to determine if extended (> 12 months) DAPT was associated with a net clinical benefit. The study population included patients who underwent successful CTO PCI within Kaiser Permanente Northern California between 2009 and 2016. Baseline demographic, clinical, and procedural characteristics were compared for patients on DAPT ≤ versus > 12 months. Clinical outcomes (death, myocardial infarction (MI), and ≥ Academic Research Consortium type 3a bleeding) were compared beginning 12 months after PCI using Cox proportional hazards models. We also adjudicated individual causes of death. 1,069 patients were followed for a median of 3.6 years (Interquartile Range = 2.2 to 5.5) following CTO PCI. Patients on DAPT ≤ 12 months (n = 597, 56%) were more likely to have anemia, end stage renal disease, and previous MI. After adjustment for between group differences, > 12 months of DAPT was associated with lower death or MI (hazard ratio [HR]: 0.66; 95% confidence interval [CI]: 0.47 to 0.93) and lower death (HR: 0.54; 95% CI: 0.36 to 0.82). There were no associations with MI (HR: 0.91; 95% CI: 0.55 to 1.5) or bleeding (HR 1.1; 95% CI: 0.50 to 2.4), but a numerically higher proportion of patients on shorter v. longer DAPT died of a cardiovascular cause (37% vs 20%, p = 0.10). In conclusion, > 12 months of DAPT was associated with lower death or MI, without an increase in bleeding. Prospective studies are needed to evaluate the optimal duration of DAPT in this unique subgroup.

Rickettsioses in Children - A Review.

Rickettsial diseases, caused by a variety of obligate intracellular, Gram-negative bacteria from the genera Rickettsia, Orientia, Ehrlichia, Neorickettsia, Neoehrlichia, and Anaplasma are considered some of the most covert emerging and re-emerging diseases. Scrub typhus, murine flea-borne typhus and Indian tick typhus are commonly being reported and during the last decade. Scrub typhus (ST) has emerged as a serious public health problem in India. Rickettsial infections are generally incapacitating and difficult to diagnose; untreated cases have case fatality rates as high as 30-45% with multiple organ dysfunction, if the specific treatment is delayed. Early clinical suspicion, timely diagnosis followed by institution of specific antimicrobial therapy shortens the course of the disease, lowers the risk of complications and reduces morbidity and mortality due to rickettsial diseases. Still there is large gap in our knowledge of Rickettsioses and the vast variability and non-specific presentation of these have often made it difficult to diagnose clinically. The present review describes the epidemiology, clinical manifestations, diagnostic modalities and treatment of Scrub typhus which is a vastly underdiagnosed entity and clinicians should suspect and test for the disease more often.

Fluorescent amino acids as versatile building blocks for chemical biology.

Fluorophores have transformed the way we study biological systems, enabling non-invasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein-protein interactions in situ or imaging nanoscopic events in real time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of fluorescent amino acids and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using site-specific genetic encoding and bioorthogonal approaches, and their application to design novel artificial proteins, as well as to investigate biological processes in cells by means of optical imaging.

Site-Specific Encoding of Photoactivity in Antibodies Enables Light-Mediated Antibody-Antigen Binding on Live Cells.

Antibodies have found applications in several fields, including, medicine, diagnostics, and nanotechnology, yet methods to modulate antibody-antigen binding using an external agent remain limited. Here, we have developed photoactive antibody fragments by genetic site-specific replacement of single tyrosine residues with photocaged tyrosine, in an antibody fragment, 7D12. A simple and robust assay is adopted to evaluate the light-mediated binding of 7D12 mutants to its target, epidermal growth factor receptor (EGFR), on the surface of cancer cells. Presence of photocaged tyrosine reduces 7D12-EGFR binding affinity by over 20-fold in two out of three 7D12 mutants studied, and binding is restored upon exposure to 365 nm light. Molecular dynamics simulations explain the difference in effect of photocaging on 7D12-EGFR interaction among the mutants. Finally, we demonstrate the application of photoactive antibodies in delivering fluorophores to EGFR-positive live cancer cells in a light-dependent manner.

Prevalence of Pediatric Metabolic Syndrome and Associated Risk Factors among School-Age Children of 10-16 Years Living in District Shimla, Himachal Pradesh, India.

INTRODUCTION: Recently, an increasing trend in the prevalence of pediatric metabolic syndrome (PMS) among school-age children has been documented in different parts of India. There is lack of data on the prevalence of PMS and its associated risk factors among school-age children living in district Shimla, Himachal Pradesh. Hence, to fill in the gap in the existing knowledge, the present study was conducted. METHODOLOGY: A cross-sectional study was conducted during 2015-2016. Thirty clusters (schools) were identified from a list of all schools using population proportionate to size sampling methodology. From each school, 70 children in the age group of 10-16 years were selected. Data was collected on the sociodemographic characteristics, anthropometry, waist circumference, blood pressure, and physical activity. Fasting venous blood samples were collected for estimation of blood glucose, triglycerides, and high-density lipoprotein levels. RESULTS: The prevalence of PMS using International Diabetes Federation classification was 3.3% and using modified-adult treatment panel classification criteria was 3.5%. Risk factors identified to be associated with PMS among school-age children were (i) male gender, (ii) high family monthly income, (iii) sedentary lifestyle, (iv) consumption of evening snack, (v) television/computer viewing, and (vi) motorized transportation for commuting to school. CONCLUSION: The PMS prevalence was 3.3% in school-age children residing in District Shimla. There is a need to formulate interventions to prevent and correct metabolic syndrome among them for reducing early onset of cardiovascular disease during adulthood.

Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog.

Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access--including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.

DNA-catalyzed lysine side chain modification.

Catalyzing the covalent modification of aliphatic amino groups, such as the lysine (Lys) side chain, by nucleic acids has been challenging to achieve. Such catalysis will be valuable, for example, for the practical preparation of Lys-modified proteins. We previously reported the DNA-catalyzed modification of the tyrosine and serine hydroxy side chains, but Lys modification has been elusive. Herein, we show that increasing the reactivity of the electrophilic reaction partner by using 5'-phosphorimidazolide (5'-Imp) rather than 5'-triphosphate (5'-ppp) enables the DNA-catalyzed modification of Lys in a DNA-anchored peptide substrate. The DNA-catalyzed reaction of Lys with 5'-Imp is observed in an architecture in which the nucleophile and electrophile are not preorganized. In contrast, previous efforts showed that catalysis was not observed when Lys and 5'-ppp were used in a preorganized arrangement. Therefore, substrate reactivity is more important than preorganization in this context. These findings will assist ongoing efforts to identify DNA catalysts for reactions of protein substrates at lysine side chains.

Concerted, rapid, quantitative, and site-specific dual labeling of proteins.

Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.

Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal.

Identifying the proteins synthesized at specific times in cells of interest in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.

Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET.

The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer.