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Abstract Development of an artificial cornea can potentially fulfil the demand of donor corneas for transplantation as the number of donors is far less than needed to treat corneal blindness. Collagen-based artificial corneas stand out as a regenerative option, having promising clinical outcomes. Collagen crosslinked with chemical crosslinkers which modify the parent functional groups of collagen. However, crosslinkers are usually cytotoxic, so crosslinkers need to be removed from implants completely before application in humans. In addition, crosslinked products are mechanically weak and susceptible to enzymatic degradation. We developed a crosslinker free supramolecular gelation strategy using pyrene conjugated dipeptide amphiphile (PyKC) consisting of lysine and cysteine; in which collagen molecules are intertwined inside the PyKC network without any functional group modification of the collagen. The newly developed collagen implants (Coll-PyKC) are optically transparent and can effectively block UV light, are mechanically and enzymatically stable, and can be sutured. The Coll-PyKC implants support the growth and function of all corneal cells, trigger anti-inflammatory differentiation while suppressing the pro-inflammatory differentiation of human monocytes. Coll-PyKC implants can restrict human adenovirus propagation. Therefore, this crosslinker-free strategy can be used for the repair, healing, and regeneration of the cornea, and potentially other damaged organs of the body.

Alphaviruses are serious zoonotic threats responsible for significant morbidity, causing arthritis or encephalitis. So far, no licensed drugs or vaccines are available to combat alphaviral infections. About 300,000 chikungunya virus (CHIKV) infections have been reported in 2023, with more than 300 deaths, including reports of a few cases in the USA as well. The discovery and development of small-molecule drugs have been revolutionized over the last decade. Here, we employed a cell-based screening approach using a series of in-house small-molecule libraries to test for their ability to inhibit CHIKV replication. DCR 137, a quinazoline derivative, was found to be the most potent inhibitor of CHIKV replication in our screening assay. Both, the cytopathic effect, and immunofluorescence of infected cells were reduced in a dose-dependent manner with DCR 137 post-treatment. Most importantly, DCR 137 was more protective than the traditional ribavirin drug and reduced CHIKV plaque-forming units by several log units. CHIKV-E2 protein levels were also reduced in a dose-dependent manner. Further, DCR 137 was probed for its antiviral activity against another alphavirus, the Ross River virus, which revealed effective inhibition of viral replication. These results led to the identification of a potential quinazoline candidate for future optimization that might act as a pan-alphavirus inhibitor.

As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386-563 and 386-510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.

India has the largest area of rainfed dryland agriculture globally, with a variety of distinct types of farming systems producing most of its coarse cereals, food legumes, minor millets, and large amounts of livestock. All these are vital for national and regional food and nutritional security. Yet, the rainfed drylands have been relatively neglected in mainstream agricultural and rural development policy. As a result, significant social-ecological challenges overlap in these landscapes: endemic poverty, malnutrition and land degradation. Sustainable intensification of dryland agriculture is essential for helping to address these challenges, particularly in the context of accelerating climate change. In this paper, we present 100 questions that point to the most important knowledge gaps and research priorities. If addressed, these would facilitate and inform sustainable intensification in Indian rainfed drylands, leading to improved agricultural production and enhanced ecosystem services. The horizon scanning method used to produce these questions brought together experts and practitioners involved in a broad range of disciplines and sectors. This exercise resulted in a consolidated set of questions covering the agricultural drylands, organized into 13 themes. Together, these represent a collective programme for new cross- and multi-disciplinary research on sustainable intensification in the Indian rainfed drylands.

Petrochemical wastewater poses environmental challenge globally due to the presence of multi-pollutants which are predominantly refractory in nature. Thirty-one indigenous bacterial strains belonging to Firmicutes and Proteobacteria were isolated from the wastewater of an effluent treatment plant (ETP) of a large liquid petrochemical cargo terminal located in Vishakhapatnam, India. Based on polyphasic taxonomy, 16S rDNA sequencing and phylogeny, it was found that many of the isolates were represented by petroleum hydrocarbon degrading taxa including Vibrio, Staphylococcus, Aerococcus, Acinetobacter and Exiguobacterium and some of them are potentially new species. Besides, majority of them exhibited metabolic ability to produce key enzymes linked to hydrocarbon degradation such as amylase, protease and lipase producers. Out of 31 isolates, 14 isolates showed growth in presence of toluene as the sole carbon source and most of these isolates were represented by the genus Bacillus. The bacterial isolates formed the basis of a consortium represented by 1:1 proportion and exhibited ability to reduce biological oxygen demand of the petrochemical wastewater by 88% under immobilized condition in laboratory. Response surface methodology-based optimization revealed a reduction of 93.33% biological oxygen demand within 12 h from simulated wastewater at 37°C which had a starting concentration of 400 mg L−1 of toluene. Upon scale-up from 5 L to 12 m3 per day processing capacity, approximately 92% removal of biological oxygen demand could be achieved within 18 to 20 h at the ambient condition in two moving bed biofilm reactors combined in series. The treated water attained discharge level as per the norms of Environmental Protection Agency. The bacterial biofilm that made up the consortium was found to be robust for all the pollutants handled at the ETP and also performed efficiently over the years. The technology could be developed into a sludge free system and thus can produce clear and dischargeable- grade treated water. The treated water could be recovered for reuse in land irrigation and firefighting and overall can lead to significant improvement of ecosystems.

Background. The framework currently used for living kidney donor selection is based on estimation of acceptable donor risk, under the premise that benefits are only experienced by the recipient. However, some interdependent donors might experience tangible benefits from donation that cannot be considered in the current framework (ie, benefits experienced directly by the donor that improve their daily life, well-being, or livelihood). Methods. We conducted semistructured interviews with 56 living kidney donors regarding benefits experienced from donation. Using a qualitative descriptive and constant comparative approach, themes were derived inductively from interview transcripts by 2 independent coders; differences in coding were reconciled by consensus. Results. Of 56 participants, 30 were in interdependent relationships with their recipients (shared household and/or significant caregiving responsibilities). Tangible benefits identified by participants fell into 3 major categories: health and wellness benefits, time and financial benefits, and interpersonal benefits. Participants described motivations to donate a kidney based on a more nuanced understanding of the benefits of donation than accounted for by the current “acceptable risk” paradigm. Discussion. Tangible benefits for interdependent donors may shift the “acceptable risk” paradigm (where no benefit is assumed) of kidney donor evaluation to a risk/benefit paradigm more consistent with other surgical decision-making.

With the identification of novel cAMP binding effector molecules in Trypanosoma, the role of cAMP in kinetoplastida parasites gained an intriguing breakthrough. Despite earlier demonstrations of the role of cAMP in the survival of Leishmania during macrophage infection, there is essential need to specifically clarify the involvement of cAMP in various cellular processes in the parasite. In this context, we sought to gain a comprehensive understanding of the effect of cAMP analogs and cAMP-cyclic nucleotide phosphodiesterase (PDE) inhibitors on proliferation of log phase parasites. Administration of both hydrolyzable (8-pCPT-cAMP) and nonhydrolyzable analogs (Sp-8-pCPT-cAMPS) of cAMP resulted in a significant decrease of Leishmania proliferation. Among the various PDE inhibitors, etazolate was found to be potently antiproliferative. BrdU cell proliferation and K/N/F-enumeration microscopic study revealed that both cAMP analogs and selective PDE inhibitors resulted in significant cell cycle arrest at G1 phase with reduced S-phase population. Furthermore, careful examination of the flagellar motility patterns revealed significantly reduced coordinated forward flagellar movement of the promastigotes with a concomitant decrease in cellular ATP levels. Alongside, 8-pCPT-cAMP and PDE inhibitors etazolate and trequinsin showed marked reduction in mitochondrial membrane potential. Treatment of etazolate at subcytotoxic concentration to infected macrophages significantly reduced parasite burden, and administration of etazolate to Leishmania-infected BALB/c mice showed reduced liver and spleen parasite burden. Collectively, these results imply involvement of cAMP in various crucial processes paving the avenue for developing potent antileishmanial agent.

Notable among the many communicable agents known to infect the human cornea is the human adenovirus, with less than ten adenoviruses having corneal tropism out of more than 100 known types. The syndrome of epidemic keratoconjunctivitis (EKC), caused principally by human adenovirus, presents acutely with epithelial keratitis, and later with stromal keratitis that can be chronic and recurrent. In this review, we discuss the current state of knowledge regarding the molecular biology of adenovirus infection of corneal stromal cells, among which the fibroblast-like keratocyte is the most predominant, in order to elucidate basic pathophysiologic mechanisms of stromal keratitis in the human patient with EKC.

Human adenoviruses cause disease at multiple mucosal sites, including the respiratory, gastrointestinal, and genitourinary tracts, and are common agents of conjunctivitis. One site of infection that has received sparse attention is the cornea, a transparent tissue and the window of the eye. While most adenovirus infections are self-limited, corneal inflammation (keratitis) due to adenovirus can persist or recur for months to years after infection, leading to reduced vision, discomfort, and light sensitivity. Topical corticosteroids effectively suppress late adenovirus keratitis but are associated with vision-threatening side effects. In this short review, we summarize current knowledge on infection of the cornea by adenoviruses, including corneal epithelial cell receptors and determinants of corneal tropism. We briefly discuss mechanisms of stromal keratitis due to adenovirus infection, and review an emerging therapy to mitigate adenovirus corneal infections based on evolving knowledge of corneal epithelial receptor usage.

Human adenovirus infection of the ocular surface is associated with severe keratoconjunctivitis and the formation of subepithelial corneal infiltrates, which may persist and impair vision for months to years following infection. Long term pathology persists well beyond the resolution of viral replication, indicating that the prolonged immune response is not virus-mediated. However, it is not clear how these responses are sustained or even initiated following infection. This review discusses recent work from our laboratory and others which demonstrates different entry pathways specific to both adenovirus and cell type. These findings suggest that adenoviruses may stimulate specific pattern recognition receptors in an entry/trafficking-dependent manner, leading to distinct immune responses dependent on the virus/cell type combination. Additional work is needed to understand the specific connections between adenoviral entry and the stimulation of innate immune responses by the various cell types present on the ocular surface.

Assembly of kinetochore complexes, involving greater than one hundred proteins, is essential for chromosome segregation and genome stability. Neocentromeres, or new centromeres, occur when kinetochores assemble de novo, at DNA loci not previously associated with kinetochore proteins, and they restore chromosome segregation to chromosomes lacking a functional centromere. Neocentromeres have been observed in a number of diseases and may play an evolutionary role in adaptation or speciation. However, the consequences of neocentromere formation on chromosome missegregation rates, gene expression, and three-dimensional (3D) nuclear structure are not well understood. Here, we used Candida albicans, an organism with small, epigenetically-inherited centromeres, as a model system to study the functions of twenty different neocentromere loci along a single chromosome, chromosome 5. Comparison of neocentromere properties relative to native centromere functions revealed that all twenty neocentromeres mediated chromosome segregation, albeit to different degrees. Some neocentromeres also caused reduced levels of transcription from genes found within the neocentromere region. Furthermore, like native centromeres, neocentromeres clustered in 3D with active/functional centromeres, indicating that formation of a new centromere mediates the reorganization of 3D nuclear architecture. This demonstrates that centromere clustering depends on epigenetically defined function and not on the primary DNA sequence, and that neocentromere function is independent of its distance from the native centromere position. Together, the results show that a neocentromere can form at many loci along a chromosome and can support the assembly of a functional kinetochore that exhibits native centromere functions including chromosome segregation accuracy and centromere clustering within the nucleus.

Chikungunya virus (CHIKV) has emerged as a global health concern due to its recent spread in both old and new world. So far, no CHIKV specific drug or vaccine is licensed for human use. In this study, we report production of Chikungunya virus like particles (CHIK-VLPs) using novel yeast expression system (Pichia pastoris) and its evaluation as vaccine candidate. The gene encoding structural polyprotein of CHIKV from a recent epidemic strain was cloned into yeast expression system. The multicopy integrants were processed for expression of CHIK-VLPs. The VLPs were purified and confirmed through electron microscopic analysis for their morphological identity with CHIKV. The in vitro and in vivo evaluation of CHIK-VLPs as vaccine candidate was determined in Balb/c mice. Induction of both humoral and cellular immune response was observed with different doses of CHIK-VLPs. The humoral immune response was studied through different techniques like enzyme linked immunosorbent assay, IgG Isotyping and plaque reduction neutralization test. CHIK-VLPs were found to elicit high titer of antibodies that are able to recognize native CHIKV. Higher level of IgG2a and IgG1 subtypes was identified suggestive of balanced Th1/Th2 response. Both in vitro and in vivo neutralization activity of CHIK-VLPs antibodies was observed even with low concentration, which shows its high specificity and neutralizing activity against two different CHIKV strains. Neonatal mice receiving anti-CHIK-VLPs antibodies were protected from CHIKV challenge. Induction of cellular immune response was confirmed through higher level of TNF-α, IL-10 and substantial level of IL-2, IL-4 and IFN-γ indicating a balanced response. This is the first report, where CHIK-VLPs has been expressed by Pichia pastoris and evaluated for neutralizing activity against CHIKV. These promising results indicate the utility of CHIK-VLPs as a promising vaccine candidate against emerging CHIKV.