IDR-targeting compounds suppress HPV genome replication via disruption of phospho-BRD4 association with DNA damage response factors.

Compounds binding to the bromodomains of bromodomain and extra-terminal (BET) family proteins, particularly BRD4, are promising anticancer agents. Nevertheless, side effects and drug resistance pose significant obstacles in BET-based therapeutics development. Using high-throughput screening of a 200,000-compound library, we identified small molecules targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that inhibit phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication in HPV-containing keratinocytes. Proteomic profiling identified two DNA damage response factors-53BP1 and BARD1-crucial for differentiation-associated HPV genome amplification. pBRD4-mediated recruitment of 53BP1 and BARD1 to the HPV origin of replication occurs in a spatiotemporal and BRD4 long (BRD4-L) and short (BRD4-S) isoform-specific manner. This recruitment is disrupted by phospho-IDR-targeting compounds with little perturbation of the global transcriptome and BRD4 chromatin landscape. The discovery of these protein-protein interaction inhibitors (PPIi) not only demonstrates the feasibility of developing PPIi against phospho-IDRs but also uncovers antiviral agents targeting an epigenetic regulator essential for virus-host interaction and cancer development.

Molecular and physiologic changes in the SpaceX Inspiration4 civilian crew.

Human spaceflight has historically been managed by government agencies, such as the NASA Twins Study1, but new commercial spaceflight opportunities have opened spaceflight to a broader population. In 2021, the SpaceX Inspiration4 mission launched the first-ever all civilian crew to low Earth orbit, which included the youngest American astronaut (age 29), novel in-flight experimental technologies (handheld ultrasound imaging, smartwatch wearables, and immune profiling), ocular alignment measurements, and new protocols for in-depth, multi-omic molecular and cellular profiling. Here we report the primary findings from the 3-day spaceflight mission, which induced a broad range of physiological and stress responses, neurovestibular changes indexed by ocular misalignment, and altered neurocognitive functioning, some of which match long-term spaceflight2, but almost all of which did not differ from baseline (pre-flight) after return to Earth. Overall, these preliminary civilian spaceflight data suggest that short-duration missions do not pose a significant health risk, and moreover present a rich opportunity to measure the earliest phases of adaptation to spaceflight in the human body at anatomical, cellular, physiologic, and cognitive levels. Finally, these methods and results lay the foundation for an open, rapidly expanding biomedical database for astronauts3, which can inform countermeasure development for both private and government-sponsored space missions.

High-resolution optical microscopy for characterising microstructural deformation in microtensile testing.

Imaging surface deformation of a coupon specimen in microtensile testing with an optical microscope presents challenges due to the narrow depth of field (DoF) of optical microscopes. Materials being heterogeneous at microscopic length scale, the sample surface deforms into a complex 3D surface texture, evolving continuously as the loading increases. Because of the narrow DoF, the region that is in focus within the field of view (FoV) decreases substantially in size with the increasing out-of-plane heterogeneous deformation. To address this challenge, a method based on image blending and stabilisation of the captured image frames is proposed. Image blending combines the partial regions that are in focus from a set of successive image frames captured at different working distances from the object surface plane to construct a single image that has a large part of the FoV in focus. The blended images are then obtained at different levels of macroscopic strains, that is the global homogeneous strain, in order to characterise the evolution of the heterogeneous deformation. The image stabilisation removes any misalignments of the blended images by spatially realigning them choosing a common feature as a reference point. The validation of the proposed method with conventionally and additively manufactured stainless steel 316L (SS 316L) specimens demonstrates excellent improvement in image quality. Almost 100% of the FoV is maintained in focus regardless of the amount of out-of-plane heterogeneous deformation caused during tensile testing, which is quite remarkable for optical microscopy imaging. Consequently, the blended and stabilised images enhanced the accuracy of digital image correlation (DIC). Time-lapse videos of the deformation generated using these images captured the evolution of the slip bands and their transmission through twinning boundaries in the stainless steel microstructure. Overall, this study demonstrates the feasibility of using image-processing techniques to advance optical microscopy to image complex 3D surfaces evolving with time.

Vorinostat, a pan-HDAC inhibitor, abrogates productive HPV-18 DNA amplification.

Human papillomaviruses (HPVs) cause epithelial proliferative diseases. Persistent infection of the mucosal epithelia by the high-risk genotypes can progress to high-grade dysplasia and cancers. Viral transcription and protein activities are intimately linked to regulation by histone acetyltransferases and histone deacetylases (HDACs) that remodel chromatin and regulate gene expression. HDACs are also essential to remodel and repair replicating chromatin to enable the progression of replication forks. As such, Vorinostat (suberoylanilide hydroximic acid), and other pan-HDAC inhibitors, are used to treat lymphomas. Here, we investigated the effects of Vorinostat on productive infection of the high-risk HPV-18 in organotypic cultures of primary human keratinocytes. HPV DNA amplifies in the postmitotic, differentiated cells of squamous epithelia, in which the viral oncoproteins E7 and E6 establish a permissive milieu by destabilizing major tumor suppressors, the pRB family proteins and p53, respectively. We showed that Vorinostat significantly reduced these E6 and E7 activities, abrogated viral DNA amplification, and inhibited host DNA replication. The E7-induced DNA damage response, which is critical for both events, was also compromised. Consequently, Vorinostat exposure led to DNA damage and triggered apoptosis in HPV-infected, differentiated cells, whereas uninfected tissues were spared. Apoptosis was attributed to highly elevated proapoptotic Bim isoforms that are known to be repressed by EZH2 in a repressor complex containing HDACs. Two other HDAC inhibitors, Belinostat and Panobinostat, also inhibited viral DNA amplification and cause apoptosis. We suggest that HDAC inhibitors are promising therapeutic agents to treat benign HPV infections, abrogate progeny virus production, and hence interrupt transmission.

CBETA: First Multipass Superconducting Linear Accelerator with Energy Recovery.

Energy recovery has been achieved in a multipass linear accelerator, demonstrating a technology for more compact particle accelerators operating at higher currents and reduced energy consumption. Energy delivered to the beam during the first four passes through the accelerating structure was recovered during four subsequent decelerating passes. High-energy efficiency was achieved by the use of superconducting accelerating cavities and permanent magnets. The fixed-field alternating-gradient optical system used for the return loop successfully transported electron bunches of 42, 78, 114, and 150 MeV in a common vacuum chamber. This new kind of accelerator, an eight-pass energy recovery linac, has the potential to accelerate much higher current than existing linear accelerators while maintaining small beam dimensions and consuming much less energy per electron.

Morbidity profile of communities in Bhopal city (India) vis-à-vis distance of residence from Union Carbide India Limited plant and drinking water usage pattern.

OBJECTIVE: A cross-sectional study was undertaken to assess the prevalence of morbidities in communities residing at variable distances from the closed down insecticide manufacturing plant premises of Union Carbide India Limited (UCIL), Bhopal, India and to determine association of morbidities, if any, with their drinking water usage pattern and distance of localities from the UCIL plant. MATERIALS AND METHODS: A total of 10,827 individuals belonging to 2,184 families, residing within 0-1 km (Stratum I) and 2.5-5.0 km (Stratum II) radial distances from UCIL plant were surveyed and 9,306 of them (86%) were clinically examined. Data were analyzed to examine the association between the groups of morbidities, likely due to biological and chemical water contamination, and the distance of locality from the UCIL plant. Multiple logistic regression was used to explore the risk factors for morbidities. RESULTS: Nearly similar prevalence (25.3% in stratum I, 25.8% in stratum II) and the trend of all-cause morbidities were recorded in the two strata. While morbidities related to gastrointestinal tract system (P < 0.05), auditory system (P < 0.01), neoplasm/cancers (P < 0.01) and congenital anomalies (P < 0.01) were significantly higher in stratum I, the prevalence of hypertension (6.4% stratum II, 4.7% stratum I; P < 0.01) and diabetes mellitus (3.4% stratum II, 2.0% stratum I; P < 0.001) was found significantly higher in stratum II. No association (P > 0.05) was observed between the prevalence of morbidities, likely due to the consumption of biologically or chemically contaminated drinking water, and the distance of locality/stratum from the UCIL plant. DISCUSSION AND CONCLUSION: By and large similar pattern of morbidities were recorded in the two strata suggesting that the communities, irrespective of the distance of their residences from UCIL plant or sources of their drinking water, are equally vulnerable to various morbidities.

Chronic respiratory morbidity in the Bhopal gas disaster cohorts: a time-trend analysis of cross-sectional data (1986-2016).

OBJECTIVES: In 1984, nearly 500,000 inhabitants of Bhopal city, India, were exposed to toxic gases that leaked from a nearby pesticide manufacturing plant. In 1985, four cohorts were established to assess the long-term health impact of exposure, namely, mild, moderate, severely exposed and unexposed groups. The self-reported morbidity data of these cohorts were collected by follow-up cross-sectional surveys at regular intervals over the last 35 years. The present study aimed to analyse the long-term trend of chronic (duration of symptoms >3 months) respiratory morbidity in the four cohorts, stratified by age groups. STUDY DESIGN: The design of this study is a longitudinal analysis of cross-sectional respiratory morbidity data. METHODS: Chronic respiratory morbidity data within the cohorts were analysed at 5-year intervals (first recorded data from 1986). Based on age at the time of exposure, subjects were stratified into four age groups: children (aged <10 years), teenagers (aged ≥10 to <20 years), younger adults (aged ≥20 to <40 years) and older adults (aged ≥40 years). RESULTS: During the first decade, after exposure to the toxic gases, chronic respiratory morbidity in children and teenagers was high (up to 9.1%), which declined thereafter. Progressively increasing chronic respiratory morbidity was observed in both the younger and older adult age groups within all cohorts during the initial 5-10 years after exposure. Respiratory morbidity in both the younger and older adult age groups remained high for 15-20 years and thereafter recorded a declining trend. The highest respiratory morbidity observed during this study in the younger and older adult age groups was 38.6% and 59.5%, respectively; these values were both recorded in the severely exposed cohort. CONCLUSIONS: Exposure to toxic gases released during the Bhopal gas disaster has resulted in chronic respiratory morbidity of the exposed population; this morbidity has continued over decades. The age of the individuals at the time of exposure and exposure severity were crucial determinants of the long-term trend of respiratory morbidity.

Targeting DNA Damage Response as a Strategy to Treat HPV Infections.

Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90-99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy.

Microballoon pressure sensors for particle imaging manometry in liquid and gaseous media.

We present the fabrication and testing of engineered microballoon particles that expand and contract under external pressure changes hence serving as microscopic pressure sensors. The particles consist of 12 µm hollow flexible 0.4 µm-thick parylene-C shells with and without a coating of ultrathin Al2O3 diffusion barriers, and the changes in the particle radius are measured from the particle spectral reflectivity. The microballoons display radial pressure sensitivities of 0.64 nm psi(-1) and 0.44 nm psi(-1), respectively in agreement with theoretical estimates. The microballoon devices were used for mapping the internal pressure drop within microfluidic chips. These devices experience nearly spherical symmetry which could make them potential flow-through sensors for the augmentation of particle-based flow characterization methodologies extending today's capabilities of particle imaging velocimetry.

HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures.

Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis, two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1.

Conditioned Media From Adipose-Derived Stromal Cells Accelerates Healing in 3-Dimensional Skin Cultures.

Wound healing involves a number of factors that results in the production of a "closed" wound. Studies have shown, in animal models, acceleration of wound healing with the addition of adipose-derived stromal cells (ADSC). The cause for the positive effect which these cells have on wound healing has not been elucidated. We have previously shown that addition of ADSC to the dermal equivalent in 3-dimensional skin cultures accelerates reepithelialization. We now demonstrate that conditioned media (CM) from cultured ADSC produced a similar rate of healing. This result suggests that a feedback from the 3-dimensional epithelial cultures to ADSC was not necessary to effect the accelerated reepithelialization. Mass spectrometry of CM from ADSC and primary human fibroblasts revealed differences in secretomes, some of which might have roles in the accelerating wound healing. Thus, the use of CM has provided some preliminary information on a possible mode of action.

Holographic assessment of self-phase modulation and blooming in a thermal medium.

The use of a low-power laser beam to characterize self-phase modulation (SPM) and bubble formation during thermal blooming (TB), as well as manipulation of the bubbles, is reported. First, a low-power 633 nm laser beam is used to characterize the induced refractive index profile during SPM of a focused 514 nm pump beam in absorbing liquid media, e.g., a solution of red dye in isopropyl alcohol. The induced phase change is also characterized using digital holography via the 633 nm source as the probe and reference. During TB at higher pump powers, bubble formation occurs in the liquid. Using a modified setup, which minimizes the effects of gravity, buoyancy, and convection, stable bubbles are generated. These are characterized using in-line digital holography with the 633 nm probe beam. It is shown that the bubble size depends on exposure time of the pump and that the bubble can be steered by moving a focused low-power laser beam. Finally, possible applications of these thermally generated bubbles are discussed.

Anomalous recovery characteristics of Pd modified ZnO nanorod based acetone sensor.

Unmodified and Pd modified Zinc Oxide (ZnO) hexagonal nanorods, grown by Chemical Bath Deposition (CBD), is reported in this paper for efficient detection of acetone vapor. After details structural characterization (XRD, FESEM and AFM) the nanorod based sensors were tested in resistive mode for detection of acetone in the concentration range of - 190-3040 ppm. By Pd surface modification the optimum working temperature was brought down from 350 degrees C (unmodified) to 300 degrees C with appreciable improvement in response magnitude (90% to 99%) also. Strikingly, the recovery time, after Pd modification, became faster than the corresponding response time up to certain concentrations range (190-1530 ppm) and above this concentration (> or = 1530-3040 ppm) response time was found to be faster than recovery time which is similar to the case with unmodified nanorods (for entire concentration range). There are earlier reports on such faster recovery (compared to response), but no proper explanation was provided. In this paper we tried to explain this apparent anomaly of recovery characteristics through concentration dependent reaction rate variation following Arrhenius equations. Also a correlation between the parameters of the corresponding electrical equivalent circuit of the sensor has been established.

Human papillomavirus (HPV) E7 induces prolonged G2 following S phase reentry in differentiated human keratinocytes.

The productive program of human papillomaviruses occurs in differentiated squamous keratinocytes. We have previously shown that HPV-18 DNA amplification initiates in spinous cells in organotypic cultures of primary human keratinocytes during prolonged G(2) phase, as signified by abundant cytoplasmic cyclin B1 (Wang, H. K., Duffy, A. A., Broker, T. R., and Chow, L. T. (2009) Genes Dev. 23, 181-194). In this study, we demonstrated that the E7 protein, which induces S phase reentry in suprabasal cells by destabilizing the p130 pocket protein (Genovese, N. J., Banerjee, N. S., Broker, T. R., and Chow, L. T. (2008) J. Virol. 82, 4862-4873), also elicited extensive G(2) responses. Western blots and indirect immunofluorescence assays were used to probe for host proteins known to control G(2)/M progression. E7 expression induced cytoplasmic accumulation of cyclin B1 and cdc2 in the suprabasal cells. The elevated cdc2 had inactivating phosphorylation on Thr(14) or Tyr(15), and possibly both, due to an increase in the responsible Wee1 and Myt1 kinases. In cells that harbored cytoplasmic cyclin B1 or cdc2, there was also an accumulation of the phosphatase-inactive cdc25C phosphorylated on Ser(216), unable to activate cdc2. Moreover, E7 expression induced elevated expression of phosphorylated ATM (Ser(1981)) and the downstream phosphorylated Chk1, Chk2, and JNKs, kinases known to inactivate cdc25C. Similar results were observed in primary human keratinocyte raft cultures in which the productive program of HPV-18 took place. Collectively, this study has revealed the mechanisms by which E7 induces prolonged G(2) phase in the differentiated cells following S phase induction.

Adipose-derived stromal cells accelerate wound healing in an organotypic raft culture model.

Adipose tissue is a known reservoir of multipotent mesenchymal stem cells, which can be manipulated in culture to produce cells with different phenotypes. The goal of this study was to determine whether the addition of these multipotential cells to organotypic, human skin equivalent cultures would accelerate wound healing after laser injury. For our initial studies, we were able to obtain 3-dimensional raft cultures from adult skin explanted directly onto the dermal equivalent containing human fibroblasts with or without adipose-derived stromal cells (ADSCs). Two days after laser injury, the raft cultures of skin explants that contained ADSCs had a completely healed multilayered epidermis, whereas the control raft culture without the adipose-derived cells still had areas of injury. With this encouraging outcome, these experiments were then repeated in a raft culture system initiated from dissociated primary adult human keratinocytes on the humanized dermal equivalent. Again, the cultures containing ADSCs healed faster than the control cultures. In conclusion, these data provide support to our hypothesis that ADSCs are an excellent and readily available source of factors necessary for accelerated wound healing and tissue regeneration.

Isoniazid preventive therapy among child contacts of TB patients, India.

BACKGROUND: Isoniazid preventive therapy (IPT) for child contacts of TB patients, a globally accepted intervention, needs to be evaluated in diverse geographical regions.OBJECTIVES: To assess the extent of IPT coverage and adherence, to ascertain its sociodemographic and programmatic correlates and to explore existing constraints from service providers and beneficiaries´ perspectives.METHODS: A mixed-method study was conducted in January-June 2021 in Paschim Bardhaman District, West Bengal, India. Quantitative assessment was done among 280 child contacts of TB cases registered between January and December 2020 in all TB units in the district. Primary caregivers were interviewed using a pre-designed questionnaire. Two focus group discussions with all senior treatment supervisors of the district and in-depth interviews with 12 purposively selected caregivers of the children were undertaken. Qualitative data were analysed thematically.RESULTS: Only 48.9% (137/280) of child contacts were screened; 58.9% (165/280) were initiated on IPT and 40% (66/165) adhered to a full course. Coverage of the full 6-month IPT among total study participants was 23.6% (66/280). Household visits by health personnel and initial screening significantly predicted increased coverage. Programmatic inadequacies, poor understanding, social stigma and COVID situation were major constraints.CONCLUSION: Coverage of IPT remains unacceptably low and requires health system strengthening for effectively implementing current recommendations of TB preventive treatment.

PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro.

BACKGROUND: High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation. METHODS: Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting. RESULTS: Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10. CONCLUSION: The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.

Large distal ureteric stone with high burden urothelial cancer of the entire ureter and renal pelvis: a dual pathology.

Upper-tract urothelial cancer comprises only 3% of all urothelial cancers. Risk factors include tobacco smoking, recurrent urinary infection, urolithiasis and analgesic abuse. Urolithiasis-induced chronic inflammation leads to urothelial proliferation and eventual malignant transformation. The most common association is reported with squamous cell cancer. A 54-year man under evaluation for right flank pain was diagnosed with a large distal ureteric stone and urothelial cancer of the entire right ureter and renal pelvis. The patient underwent right nephroureterectomy and stone retrieval, with urinary bladder cuff excision and pelvic lymph node dissection. On follow-up, the patient succumbed to disease recurrence with widespread metastasis. Urothelial cancer associated with stone disease is atypical. Long-standing inflammation causing metaplastic and dysplastic changes is a possible hypothesis. Careful assessment of the malignancy should be looked for in patients with long-standing obstruction due to stone disease.

Genome-wide analysis in 756,646 individuals provides first genetic evidence that ACE2 expression influences COVID-19 risk and yields genetic risk scores predictive of severe disease.

SARS-CoV-2 enters host cells by binding angiotensin-converting enzyme 2 (ACE2). Through a genome-wide association study, we show that a rare variant (MAF = 0.3%, odds ratio 0.60, P=4.5×10-13) that down-regulates ACE2 expression reduces risk of COVID-19 disease, providing human genetics support for the hypothesis that ACE2 levels influence COVID-19 risk. Further, we show that common genetic variants define a risk score that predicts severe disease among COVID-19 cases.