Green synthesis of zinc oxide nanoparticles from Wodyetia bifurcata fruit peel extract: multifaceted potential in wound healing, antimicrobial, antioxidant, and anticancer applications.

This study focuses on the synthesis, characterization, and use of zinc oxide nanoparticles (ZnONPs) derived from W. bifurcata fruit peel extract. ZnONPs are frequently synthesized utilizing a green technique that is both cost-effective and ecologically friendly. ZnONPs were characterized utilizing analytical techniques. Ultra Violet visible (UV-Vis) spectra showed peaks at 364 nm, confirming the production of ZnONPs. Scanning Electron Microscope analysis indicated that the nanoparticles generated were spherical/agglomerated, with diameters ranging from 11 to 25 nm. FTIR spectroscopy was used to identify the particular functional groups responsible for the nanoparticles' reduction, stabilization, and capping. Phytochemical analysis of the extract revealed that flavonoids, saponins, steroids, triterpenoids, and resins were present. The antibacterial activity of W. bifurcata synthesised nanoparticles was evaluated against pathogenic bacteria. The ZnONPs antioxidant activity was assessed using DPPH assay. The in vitro cytotoxicity was assessed against prostate cancer PC3 cells. The wound healing potential was assessed by employing in vitro scratch assay and in vivo excision model in Wistar rats. Because of its environmentally benign production, low toxicity, and biocompatibility, ZnONPs exhibited potential antibacterial, antioxidant, anticancer, and wound healing activities, indicating that they could be used in cancer treatment and wound management. Further study is required to examine the fundamental mechanisms and evaluate the safety and effectiveness of the test sample in clinical situations.

Enhancing recovery from gut microbiome dysbiosis and alleviating DSS-induced colitis in mice with a consortium of rare short-chain fatty acid-producing bacteria.

The human gut microbiota is a complex community comprising hundreds of species, with a few present in high abundance and the vast majority in low abundance. The biological functions and effects of these low-abundant species on their hosts are not yet fully understood. In this study, we assembled a bacterial consortium (SC-4) consisting of B. paravirosa, C. comes, M. indica, and A. butyriciproducens, which are low-abundant, short-chain fatty acid (SCFA)-producing bacteria isolated from healthy human gut, and tested its effect on host health using germ-free and human microbiota-associated colitis mouse models. The selection also favored these four bacteria being reduced in abundance in either Ulcerative Colitis (UC) or Crohn's disease (CD) metagenome samples. Our findings demonstrate that SC-4 can colonize germ-free (GF) mice, increasing mucin thickness by activating MUC-1 and MUC-2 genes, thereby protecting GF mice from Dextran Sodium Sulfate (DSS)-induced colitis. Moreover, SC-4 aided in the recovery of human microbiota-associated mice from DSS-induced colitis, and intriguingly, its administration enhanced the alpha diversity of the gut microbiome, shifting the community composition closer to control levels. The results showed enhanced phenotypes across all measures when the mice were supplemented with inulin as a dietary fiber source alongside SC-4 administration. We also showed a functional redundancy existing in the gut microbiome, resulting in the low abundant SCFA producers acting as a form of insurance, which in turn accelerates recovery from the dysbiotic state upon the administration of SC-4. SC-4 colonization also upregulated iNOS gene expression, further supporting its ability to produce an increasing number of goblet cells. Collectively, our results provide evidence that low-abundant SCFA-producing species in the gut may offer a novel therapeutic approach to IBD.

Using Small Molecules for Targeting Heavy Metals in Neurotoxicity and Neuroinflammation.

Pharmaceutical drugs, natural toxins, industrial chemicals, and various environmental toxins negatively impact the nervous system. A significant cause of many neurodegenerative diseases is neurotoxicity. Although trace amounts of heavy metals are required for the proper functioning of several metabolic pathways, their dysregulation can cause many cellular and molecular alterations, which can enhance the risks associated with several neurodegenerative diseases. For example, high levels of heavy metals like manganese (Mn) affect the central nervous system with implications in both higher-order cognitive and motor functions. In addition, the buildup of amyloid aggregates and metal ions in the brain of patients with Alzheimer's disease is associated with disease pathogenesis. Small molecules capable of targeting neuroinflammation and neuroprotection pathways would be valuable to elucidate the pathological pathways associated with metal toxicity in neurogenerative disease. This chapter will summarize the necessary steps involved in (1) culturing of cell lines and maintenance of animal models, (2) design and preparation of samples of small molecules and treatment methodologies, (3) RNA and protein isolation and preparation of tissue and cell culture samples for quantitative studies, and (4) quantitative estimation of cellular products.

Impacts of Omega-3 Fatty Acids, Natural Elixirs for Neuronal Health, on Brain Development and Functions.

Omega-3 fatty acids play a seminal role in maintaining the structural and functional integrity of the nervous system. These specialized molecules function as precursors for many lipid-based biological messengers. Also, studies suggest the role of these fatty acids in regulating healthy sleep cycles, cognitive ability, brain development, etc. Dietary intake of essential poly unsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are foundational to the optimal working of the nervous system. Besides regulating health, these biomolecules have great therapeutic value in treating several diseases, particularly nervous system diseases and disorders. Many recent studies conclusively demonstrated the beneficial effects of Omega-3 fatty acids in treating depression, neuropsychiatric disorders, neurodegenerative disorders, neurochemical disorders, and many other illnesses associated with the nervous system. This chapter summates the multifaceted role of poly unsaturated fatty acids, especially Omega-3 fatty acids (EPA and DHA), in the neuronal health and functioning. The importance of dietary intake of these essential fatty acids, their recommended dosages, bioavailability, the mechanism of their action, and therapeutic values are extensively discussed.

Comparing the effects of packaging normal-pH and atypical dark-cutting beef in modified atmosphere conditions on surface color.

Limited studies have determined the effects of modified atmospheric packaging (MAP) on atypical dark-cutting beef surface color. The objective was to compare the impacts of using vacuum packaging, carbon monoxide (CO-MAP), and HiOx-MAP (high­oxygen) on the retail color of normal-pH and atypical dark-cutting beef aged 14 d. Atypical dark-cutting beef (pH 5.63) had numerically greater (P > 0.05) pH than normal-pH beef (pH 5.56). Atypical dark-cutting steaks were darker in color (lower L* values; P < 0.05) than normal-pH steaks. Atypical dark-cutting steaks had greater (P < 0.05) oxygen consumption, lower (P < 0.05) relative oxygenation, and less inter-muscle bundle space (P < 0.05) than normal-pH steaks. There were no differences (P > 0.05) in redness between normal-pH and atypical dark-cutting steaks when packaged in HiOx-MAP. Although a minimal increase in pH was observed in atypical dark-cutting beef, steaks in CO-MAP had lower redness than normal-pH steaks.

Acellular Human Amniotic Fluid-Derived Extracellular Vesicles as Novel Anti-Inflammatory Therapeutics against SARS-CoV-2 Infection.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 is associated with acute respiratory distress syndrome (ARDS) and fatal pneumonia. Excessive inflammation caused by SARS-CoV-2 is the key driver of ARDS and lethal disease. Several FDA-approved drugs that suppress virus replication are in clinical use. However, despite strong evidence for the role of virus-induced inflammation in severe COVID-19, no effective anti-inflammatory drug is available to control fatal inflammation as well as efficiently clear the virus. Therefore, there is an urgent need to identify biologically derived immunomodulators that suppress inflammation and promote antiviral immunity. In this study, we evaluated acellular human amniotic fluid (acAF) containing extracellular vesicles (hAF-EVs) as a potential non-toxic and safe biologic for immunomodulation during COVID-19. Our in vitro results showed that acAF significantly reduced inflammatory cytokine production in TLR2/4/7 and SARS-CoV-2 structural protein-stimulated mouse macrophages. Importantly, an intraperitoneal administration of acAF reduced morbidity and mortality in SARS-CoV-2-infected mice. A detailed examination of SARS-CoV-2-infected lungs revealed that the increased protection in acAF-treated mice was associated with reduced viral titers and levels of inflammatory myeloid cell infiltration. Collectively, our results identify a novel biologic that has potential to suppress excessive inflammation and enhance survival following SARS-CoV-2 infection, highlighting the translational potential of acAF against COVID-19.

Identification of a microbial sub-community from the feral chicken gut that reduces Salmonella colonization and improves gut health in a gnotobiotic chicken model.

A complex microbial community in the gut may prevent the colonization of enteric pathogens such as Salmonella. Some individual or a combination of species in the gut may confer colonization resistance against Salmonella. To gain a better understanding of the colonization resistance against Salmonella enterica, we isolated a library of 1,300 bacterial strains from feral chicken gut microbiota which represented a total of 51 species. Using a co-culture assay, we screened the representative species from this library and identified 30 species that inhibited Salmonella enterica subspecies enterica serovar Typhimurium in vitro. To improve the Salmonella inhibition capacity, from a pool of fast-growing species, we formulated 66 bacterial blends, each of which composed of 10 species. Bacterial blends were more efficient in inhibiting Salmonella as compared to individual species. The blend that showed maximum inhibition (Mix10) also inhibited other serotypes of Salmonella frequently found in poultry. The in vivo effect of Mix10 was examined in a gnotobiotic and conventional chicken model. The Mix10 consortium significantly reduced Salmonella load at day 2 post-infection in gnotobiotic chicken model and decreased intestinal tissue damage and inflammation in both models. Cell-free supernatant of Mix10 did not show Salmonella inhibition, indicating that Mix10 inhibits Salmonella through either nutritional competition, competitive exclusion, or through reinforcement of host immunity. Out of 10 species, 3 species in Mix10 did not colonize, while 3 species constituted more than 70% of the community. Two of these species were previously uncultured bacteria. Our approach could be used as a high-throughput screening system to identify additional bacterial sub-communities that confer colonization resistance against enteric pathogens and its effect on the host.IMPORTANCESalmonella colonization in chicken and human infections originating from Salmonella-contaminated poultry is a significant problem. Poultry has been identified as the most common food linked to enteric pathogen outbreaks in the United States. Since multi-drug-resistant Salmonella often colonize chicken and cause human infections, methods to control Salmonella colonization in poultry are needed. The method we describe here could form the basis of developing gut microbiota-derived bacterial blends as a microbial ecosystem therapeutic against Salmonella.

Mutation of a highly conserved isoleucine residue in loop 2 of several ß-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental to infection.

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication.

Thermoalkalophilic polygalacturonase from a novel Glutamicibacter sp.: Bioprospecting, strain improvement, statistical optimization and applications.

The current research discusses a multidimensional bioprocess development, that includes bioprospecting, strain improvement, media optimisation, and applications of the extracted enzyme. A potent alkalophilic polygalacturonase (PG) producing bacterial strain was isolated and identified as a novel Glutamicibacter sp. Furthermore, strain improvement by UV and chemical mutagenesis not only improved the enzyme (PGmut) production but also enhanced its temperature optima from 37 °C to 50 °C. The use of solid substrate fermentation, followed bystatistical optimisation through PB and RSM, substantially increasedPGmut production. A 10-fold increase in enzyme production (632 U/gm) was observed when sugarcane bagasse with a pH of 10.5, 66.8 % moisture, and an inoculum size of 10.15 % was used. The model's accuracy was supported by p-value (p < 0.0001), and an R2 of 0.9940. A pilot-scale experiment, demonstrated ≈ 62,229 U/100 gm PG activity. Additionally, the enzyme's efficacy in demucilization of coffee beans, and bioscouring of jute fibre indicated that it is a valuable biocatalyst.

N-dihydrogalactochitosan serves as an effective mucosal adjuvant for intranasal vaccine in combination with recombinant viral proteins against respiratory infection.

Mucosal vaccinations for respiratory pathogens provide effective protection as they stimulate localized cellular and humoral immunities at the site of infection. Currently, the major limitation of intranasal vaccination is using effective adjuvants capable of withstanding the harsh environment imposed by the mucosa. Herein, we describe the efficacy of using a unique biopolymer, N-dihydrogalactochitosan (GC), as a nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV), an MF-59 equivalent. In contrast to AV, intranasal application of GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. Moreover, GC+S+NC-vaccinated animals were largely resistant to the lethal SARS-CoV-2 challenge and experienced drastically reduced morbidity and mortality, with animal weights and behavior returning to normal 22 days post-infection. In contrast, animals intranasally vaccinated with AV+S+NC experienced severe weight loss, mortality, and respiratory distress, with none surviving beyond 6 days post-infection. Our findings demonstrate that GC can serve as a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses. STATEMENT OF SIGNIFICANCE: We demonstrated that a unique biopolymer, N-dihydrogalactochitosan (GC), was an effective nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV). In contrast to AV, GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. About 90 % of the GC+S+NC-vaccinated animals survived the lethal SARS-CoV-2 challenge and remained healthy 22 days post-infection, while the AV+S+NC-vaccinated animals experienced severe weight loss and respiratory distress, and all died within 6 days post-infection. Our findings demonstrate that GC is a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses.

PARP12 is required to repress the replication of a Mac1 mutant coronavirus in a cell- and tissue-specific manner.

ADP-ribosyltransferases (ARTs) mediate the transfer of ADP-ribose from NAD+ to protein or nucleic acid substrates. This modification can be removed by several different types of proteins, including macrodomains. Several ARTs, also known as PARPs, are stimulated by interferon indicating ADP-ribosylation is an important aspect of the innate immune response. All coronaviruses (CoVs) encode for a highly conserved macrodomain (Mac1) that is critical for CoVs to replicate and cause disease, indicating that ADP-ribosylation can effectively control coronavirus infection. Our siRNA screen indicated that PARP12 might inhibit the replication of a murine hepatitis virus (MHV) Mac1 mutant virus in bone-marrow-derived macrophages (BMDMs). To conclusively demonstrate that PARP12 is a key mediator of the antiviral response to CoVs both in cell culture and in vivo, we produced PARP12-/-mice and tested the ability of MHV A59 (hepatotropic/neurotropic) and JHM (neurotropic) Mac1 mutant viruses to replicate and cause disease in these mice. Notably, in the absence of PARP12, Mac1 mutant replication was increased in BMDMs and mice. In addition, liver pathology was also increased in A59-infected mice. However, the PARP12 knockout did not restore Mac1 mutant virus replication to WT virus levels in all cell or tissue types and did not significantly increase the lethality of Mac1 mutant viruses. These results demonstrate that while PARP12 inhibits MHV Mac1 mutant virus infection, additional PARPs or innate immune factors must contribute to the extreme attenuation of this virus in mice. IMPORTANCE Over the last decade, the importance of ADP-ribosyltransferases (ARTs), also known as PARPs, in the antiviral response has gained increased significance as several were shown to either restrict virus replication or impact innate immune responses. However, there are few studies showing ART-mediated inhibition of virus replication or pathogenesis in animal models. We found that the CoV macrodomain (Mac1) was required to prevent ART-mediated inhibition of virus replication in cell culture. Using knockout mice, we found that PARP12, an interferon-stimulated ART, was required to repress the replication of a Mac1 mutant CoV both in cell culture and in mice, demonstrating that PARP12 represses coronavirus replication. However, the deletion of PARP12 did not fully rescue Mac1 mutant virus replication or pathogenesis, indicating that multiple PARPs function to counter coronavirus infection.

Klebsiella pneumoniae C o-infection Leads to Fatal Pneumonia in SARS-CoV-2-infected Mice.

SARS-CoV-2 patients have been reported to have high rates of secondary Klebsiella pneumoniae infections. Klebsiella pneumoniae is a commensal that is typically found in the respiratory and gastrointestinal tracts. However, it can cause severe disease when a person's immune system is compromised. Despite a high number of K. pneumoniae cases reported in SARS-CoV-2 patients, a co-infection animal model evaluating the pathogenesis is not available. We describe a mouse model to study disease pathogenesis of SARS-CoV-2 and K. pneumoniae co-infection. BALB/cJ mice were inoculated with mouse-adapted SARS-CoV-2 followed by a challenge with K. pneumoniae . Mice were monitored for body weight change, clinical signs, and survival during infection. The bacterial load, viral titers, immune cell accumulation and phenotype, and histopathology were evaluated in the lungs. The co-infected mice showed severe clinical disease and a higher mortality rate within 48 h of K. pneumoniae infection. The co-infected mice had significantly elevated bacterial load in the lungs, however, viral loads were similar between co-infected and single-infected mice. Histopathology of co-infected mice showed severe bronchointerstitial pneumonia with copious intralesional bacteria. Flow cytometry analysis showed significantly higher numbers of neutrophils and macrophages in the lungs. Collectively, our results demonstrated that co-infection of SARS-CoV-2 with K. pneumoniae causes severe disease with increased mortality in mice.

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice.

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.

Treating methicillin-resistant Staphylococcus aureus (MRSA) bone infection with focused ultrasound combined thermally sensitive liposomes.

OBJECTIVE: Chronic bone infection caused by Staphylococcus aureus biofilms in children and adults is characterized by reduced antibiotic sensitivity. In this study, we assessed 'heat-targeted, on-demand' antibiotic delivery for S. aureus killing by combining ciprofloxacin (CIP)-laden low-temperature sensitive liposomes (LTSLs) with local high-intensity focused ultrasound (HIFU) induced bone heating in a rat model of bone infection. METHODS: CIP-LTSLs were prepared using the thin-film hydration and extrusion method. Bone infection was established by surgically implanting an orthopedic K-wire colonized with methicillin-resistant S. aureus (MRSA) strain into rat's femurs. For bone heating, ultrasound-guided HIFU exposures were performed to achieve a local temperature of 40-42 °C (∼15 min) concurrently with intravenous injection of CIP-LTSLs or CIP. CIP biodistribution was determined spectrophotometrically and therapeutic efficacy was determined by bacteriological, histological and scanning electron microscopy (SEM) analyses. RESULTS: CIP-LTSLs in the range of 183.5 nm ± 1.91 showed an encapsulation efficiency of >70% at 37 °C and a complete release at ∼42 °C. The metal implantation method yielded medullary osteomyelitis characterized by suppurative changes (bacterial and pus pockets) by day 10 in bones and adjoining muscle tissues. HIFU heating significantly improved CIP delivery from LTSLs in bones, resulting in a significant reduction in MRSA load compared to HIFU and CIP alone groups. These were also verified by histology and SEM, wherein a distinct reduction in S. aureus population in the infected metal wires and tissues from the combinatorial therapy was noted. CONCLUSION: HIFU improved CIP delivery to bones, achieving clearance of hard-to-treat MRSA biofilms.

PARP12 is required to repress the replication of a Mac1 mutant coronavirus in a cell and tissue specific manner.

ADP-ribosyltransferases (ARTs) mediate the transfer of ADP-ribose from NAD + to protein or nucleic acid substrates. This modification can be removed by several different types of proteins, including macrodomains. Several ARTs, also known as PARPs, are stimulated by interferon, indicating ADP-ribosylation is an important aspect of the innate immune response. All coronaviruses (CoVs) encode for a highly conserved macrodomain (Mac1) that is critical for CoVs to replicate and cause disease, indicating that ADP-ribosylation can effectively control coronavirus infection. Our siRNA screen indicated that PARP12 might inhibit the replication of a MHV Mac1 mutant virus in bone-marrow derived macrophages (BMDMs). To conclusively demonstrate that PARP12 is a key mediator of the antiviral response to CoVs both in cell culture and in vivo , we produced PARP12 -/- mice and tested the ability of MHV A59 (hepatotropic/neurotropic) and JHM (neurotropic) Mac1 mutant viruses to replicate and cause disease in these mice. Notably, in the absence of PARP12, Mac1 mutant replication was increased in BMDMs and in mice. In addition, liver pathology was also increased in A59 infected mice. However, the PARP12 knockout did not restore Mac1 mutant virus replication to WT virus levels in all cell or tissue types and did not significantly increase the lethality of Mac1 mutant viruses. These results demonstrate that while PARP12 inhibits MHV Mac1 mutant virus infection, additional PARPs or innate immune factors must contribute to the extreme attenuation of this virus in mice. IMPORTANCE: Over the last decade, the importance of ADP-ribosyltransferases (ARTs), also known as PARPs, in the antiviral response has gained increased significance as several were shown to either restrict virus replication or impact innate immune responses. However, there are few studies showing ART-mediated inhibition of virus replication or pathogenesis in animal models. We found that the CoV macrodomain (Mac1) was required to prevent ART-mediated inhibition of virus replication in cell culture. Here, using knockout mice, we found that PARP12, an interferon-stimulated ART, was required to repress the replication of a Mac1 mutant CoV both in cell culture and in mice, demonstrating that PARP12 represses coronavirus replication. However, the deletion of PARP12 did not fully rescue Mac1 mutant virus replication or pathogenesis, indicating that multiple PARPs function to counter coronavirus infection.

Statistical bioprocess optimization for enhanced production of a thermo alkalophilic polygalacturonase (PGase) from Pseudomonas sp. 13156349 using solid substrate fermentation (SSF).

In this study, a polygalacturonase (PGase) producing bacterial strain was isolated and identified as Pseudomonas sp. 13159349 from fruit market soils, and TLC analysis confirmed its pectinolytic activity. Additionally, SSF, Plackett-Burman design (PB), and response surface methodology (RSM) were used to optimize the production of this thermostable and alkalophilic PGase. Wheat bran demonstrated the highest activity (60.13 ± 3.39 U/gm) among the various agricultural wastes used as solid substrates. To further enhance the enzyme production, statistical optimization of media components was investigated using the PB design. Among the 11 variables tested, pH (p < 0.0001), inoculum size (p < 0.0001), incubation time (p < 0.0001), and temperature (p < 0.0041) were found to have a positive effect on the production. The interaction and concentration of the selected factors were examined by RSM, which demonstrated the optimal conditions for maximum production (315.65 U/gm) of the enzyme using wheat bran as the solid substrate were pH 10.5, 61-66 h of incubation, and 6-7.5% inoculum size. The model was highly significant, with a p-value of <0.0001, an F-value of 95.33, and a low CV of 2.31. The RSM model was validated by a laboratory-scale experiment showing 30600 ± 400.32 U/100 gm PGase activity. Thus, SSF and the statistical design of media components resulted in a significant 5.2-fold increase in PGase output solely by using agro waste and optimizing the physical parameters, making this a highly cost-effective bioprocess.

Enabling Chemo-Immunotherapy with HIFU in Canine Cancer Patients.

High intensity focused ultrasound (HIFU) is a promising non-invasive technique for treating solid tumors using thermal and histotripsy-based mechanical ablation. However, its clinical significance in different tumor types is not fully understood. To assess its therapeutic efficacy and immunomodulatory properties, we compared HIFU thermal ablation and histotripsy ablation in dogs with spontaneous tumors. We also evaluated the ability of non-ablative HIFU-based mild hyperthermia (40-45 ºC) to improve Doxorubicin delivery and immunomodulation. Our results showed that HIFU thermal ablation induced tumor remission in the majority of treated patients over 60 days, while histotripsy achieved partial response to stable disease persistence. The adverse effects of thermal ablation were minor to moderate, while histotripsy exposures were relatively well-tolerated. Furthermore, we observed a correlation between HIFU-therapeutic response and serum anti-tumor cytokine profiles and the presence of functionally active cytotoxic immune cells in patients. Similarly, Doxorubicin-treated patients showed improved drug delivery, efficacy, and anti-tumor immune responses with HIFU hyperthermia. In conclusion, our study demonstrates that depending on the tumor type and treatment parameters, HIFU treatments can enable tumor growth control, immune activation, and chemotherapy in veterinary patient. These findings have significant clinical implications and highlight the potential of HIFU as a promising cancer treatment approach.

Toll-like receptor 7 (TLR7)-mediated antiviral response protects mice from lethal SARS-CoV-2 infection.

SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses cause fatal pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and lethal inflammatory responses in-vivo are not well defined. CoVs possess single-stranded RNA (ssRNA) genome that is abundantly produced during infection and stimulates both antiviral interferon (IFN) and inflammatory cytokine/ chemokine responses. Therefore, in this study, using wild-type control and TLR7 deficient BALB/c mice infected with a mouse-adapted SARS-COV-2 (MA-CoV-2), we evaluated the role of TLR7 signaling in MA-CoV-2-induced antiviral and inflammatory responses and disease outcome. We show that TLR7-deficient mice are more susceptible to MA-CoV-2 infection as compared to infected control mice. Further evaluation of MA-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral type I (IFNα/ß) and type III (IFNλ) IFNs, IFN stimulated genes (ISGs, ISG15 and CXCL10), and several pro-inflammatory cytokines/chemokines in TLR7 deficient compared to control mice. Reduced lung IFN/ISG levels and increased morbidity/mortality in TLR7 deficient mice correlated with high lung viral titer. Detailed examination of total cells from MA-CoV-2 infected lungs showed high neutrophil count in TLR7 deficient mice compared to control mice. Additionally, blocking TLR7 activity post-MA-CoV-2 infection using a specific inhibitor also enhanced disease severity. In summary, our results conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite robust inflammatory response, TLR7-mediated IFN/ISG responses likely protect the host from lethal disease. Given similar outcomes in control and TLR7 deficient humans and mice, these results show that MA-CoV-2 infected mice serve as excellent model to study COVID-19.

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in mice.

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in this set of proteins is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and interferon-stimulated gene (ISG) expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target. SIGNIFICANCE: All CoVs, including SARS-CoV-2, encode for a conserved macrodomain (Mac1) that counters host ADP-ribosylation. Prior studies with SARS-CoV-1 and MHV found that Mac1 blocks IFN production and promotes CoV pathogenesis, which has prompted the development of SARS-CoV-2 Mac1 inhibitors. However, development of these compounds into antivirals requires that we understand how SARS-CoV-2 lacking Mac1 replicates and causes disease in vitro and in vivo . Here we found that SARS-CoV-2 containing a complete Mac1 deletion replicates normally in cell culture but induces an elevated IFN response, has reduced viral loads in vivo , and does not cause significant disease in mice. These results will provide a roadmap for testing Mac1 inhibitors, help identify Mac1 functions, and open additional avenues for coronavirus therapies.

Influence of Ag on the Structural, Electrochemical, Antibacterial, and Photocatalytic Performance of the (CuO-Cu2O)Cu Nanocomposite.

The cost-effective novel Ag-doped (1-7%) (CuO-Cu2O)Cu (C3) heterostructured nanocomposites are successfully synthesized by the facile solution combustion process using the Leucas aspera extract as a green fuel. The structural properties of fabricated nanocomposites were well-characterized by specific spectral techniques for enhanced electrochemical sensor detection, antibacterial activities, and sunlight-driven photocatalytic dye decoloration studies. The existence of Ag+ ions has been confirmed by the appearance of two peaks of Ag 3d5/2 (367.9 eV) and Ag 3d3/2 (373.9 eV), with the chemical binding nature and exchange of the Ag+ state in the nanocomposite lattice as revealed by X-ray photoelectron spectroscopy analysis. The energy band gap value of the doped nanocomposite decreases from 2.2 to 1.8 eV, as measured by the UV-visible absorption spectral technique, hindering the recombination of electron-holes pairs by trapping e- and h+. This result supports that the C3Ag5 nanocomposite has a great potential as a sunlight photocatalyst toward the Alizarin Red (AR) dye, for which an excellent degradation activity of 98% at 180 min was achieved compared to that of the host nanocomposite (78% at 180 min). The variation of redox peak potentials of the prepared graphite nanocomposite working electrode is an effective tool for paracetamol sensing activity in 0.1 M KCl using electrochemical spectral studies. In addition, the antibacterial activities of the C3Ag5 nanocomposite against Escherichia coli and Staphylococcus aureus were successfully studied. The C3Ag5 nanocomposite exhibited a better performance than C3. The increase in activity is attributed to the presence of Ag as a dopant.