Modulation of Porcine Gut Microbiota and Microbiome: Hologenomic, Dietary, and Endogenous Factors.

Global pig production contributes to about 35% of the world's meat production and consumption [...].

Monomodular and multifunctional processive endocellulases: implications for swine nutrition and gut microbiome.

Poor efficiency of dietary fibre utilization not only limits global pork production profit margin but also adversely affects utilization of various dietary nutrients. Poor efficiency of dietary nutrient utilization further leads to excessive excretion of swine manure nutrients and results in environmental impacts of emission of major greenhouse gases (GHG), odor, nitrate leaching and surface-water eutrophication. Emission of the major GHG from intensive pork production contributes to global warming and deteriorates heat stress to pigs in tropical and sub-tropical swine production. Exogenous fibre enzymes of various microbial cellulases, hemicellulases and pectinases have been well studied and used in swine production as the non-nutritive gut modifier feed enzyme additives in the past over two decades. These research efforts have aimed to improve growth performance, nutrient utilization, intestinal fermentation as well as gut physiology, microbiome and health via complementing the porcine gut symbiotic microbial fibrolytic activities towards dietary fibre degradation. The widely reported exogenous fibre enzymes include the singular use of respective cellulases, hemicellulases and pectinases as well as their multienzyme cocktails. The currently applied exogenous fibre enzymes are largely limited by their inconsistent in vivo efficacy likely due to their less defined enzyme stability and limited biochemical property. More recently characterized monomodular, multifunctional and processive endoglucanases have the potential to be more efficaciously used as the next-generation designer fibre biocatalysts. These newly emerging multifunctional and processive endoglucanases have the potential to unleash dietary fibre sugar constituents as metabolic fuels and prebiotics, to optimize gut microbiome, to maintain gut permeability and to enhance performance in pigs under a challenged environment as well as to parallelly unlock biomass to manufacture biofuels and biomaterials.

[A case of repairing the destructive high-voltage electric burn wounds in the head, face, and neck based on the economic theory of flaps].

In September 8th, 2021, a male patient (aged 18 years) with severe destructive injuries of high-voltage electric burns in the head, face, and neck was admitted to General Hospital of Taiyuan Iron Steel (Group) Co., Ltd. Based on the economic theory of flaps, the flap donor site and transplantation method were optimized and evaluated before surgery, and then debridement of head, face, and neck wounds+removal of necrotic skull+free transplantation of super large latissimus dorsi myocutaneous flap+thin intermediate thickness skin graft transplantation from the left thigh was performed. The extra large flap donor site wound was sutured directly. This surgery reduced the adverse consequences of the flap donor site on the premise of ensure of repair effect. After operation, the patient's condition was stable, the flap and skin graft survived well, the repair effect of wound was well, the scar in the flap donor area was relatively mild, and the upper limb had no dysfunction.

Single-cycle influenza virus vaccine generates lung CD8+ Trm that cross-react against viral variants and subvert virus escape mutants.

Influenza virus-specific tissue-resident memory (Trm) CD8+ T cells located along the respiratory tract provide cross-strain protection against a breadth of influenza viruses. We show that immunization with a single-cycle influenza virus vaccine candidate (S-FLU) results in the deposition of influenza virus nucleoprotein (NP)-specific CD8+ Trm along the respiratory tract that were more cross-reactive against viral variants and less likely to drive the development of cytotoxic T lymphocyte (CTL) escape mutants, as compared to the lung memory NP-specific CD8+ T cell pool established following influenza infection. This immune profile was linked to the limited inflammatory response evoked by S-FLU vaccination, which increased TCR repertoire diversity within the memory CD8+ T cell compartment. Cumulatively, this work shows that S-FLU vaccination evokes a clonally diverse, cross-reactive memory CD8+ T cell pool, which protects against severe disease without driving the virus to rapidly evolve and escape, and thus represents an attractive vaccine for use against rapidly mutating influenza viruses.

Meta-analysis of the diagnostic value of serum procalcitonin for burn sepsis in adults.

OBJECTIVE: Serum procalcitonin (PCT) reflects the infection status of the organism and the severity of the infection. The purpose of this study was to systematically evaluate the diagnostic value of serum PCT for burn sepsis in adults and to provide a factual basis for future clinical diagnosis and decision-making. MATERIALS AND METHODS: On August 16, 2022, six databases were searched in this study and a total of 856 studies were found. The retrieved literature was comprehensively evaluated according to the inclusion and exclusion criteria, and the valid data were extracted and included for analysis. The number of true positives, false positives, true negatives and false negatives were used as indicators to evaluate the diagnostic value of serum PCT for burn sepsis in adults. RESULTS: In total, 15 studies met the inclusion criteria. Meta-analysis showed a combined sensitivity of 0.78 (95% CI: 0.69-0.84), a combined specificity of 0.85 (95% CI: 0.77-0.91), a combined positive likelihood ratio of 5.17 (95% CI: 3.25-8.25), a combined negative likelihood ratio of 0.26 (95% CI: 0.19-0.37), and a combined diagnostic ratio of 19.63 (95% CI: 10.17-37.90). The AUC was 0.88 (95% CI: 0.85-0.90). CONCLUSIONS: Serum PCT provides good early diagnostic benefits for burn sepsis in adults. More high-quality studies are required to clarify its specific early diagnostic value.

Deglycosylation Differentially Regulates Weaned Porcine Gut Alkaline Phosphatase Isoform Functionality along the Longitudinal Axis.

Gut alkaline phosphatases (AP) dephosphorylate the lipid moiety of endotoxin and other pathogen-associated-molecular patterns members, thus maintaining gut eubiosis and preventing metabolic endotoxemia. Early weaned pigs experience gut dysbiosis, enteric diseases and growth retardation in association with decreased intestinal AP functionality. However, the role of glycosylation in modulation of the weaned porcine gut AP functionality is unclear. Herein three different research approaches were taken to investigate how deglycosylation affected weaned porcine gut AP activity kinetics. In the first approach, weaned porcine jejunal AP isoform (IAP) was fractionated by the fast protein-liquid chromatography and purified IAP fractions were kinetically characterized to be the higher-affinity and lower-capacity glycosylated mature IAP (p < 0.05) in comparison with the lower-affinity and higher-capacity non-glycosylated pre-mature IAP. The second approach enzyme activity kinetic analyses showed that N-deglycosylation of AP by the peptide N-glycosidase-F enzyme reduced (p < 0.05) the IAP maximal activity in the jejunum and ileum and decreased AP affinity (p < 0.05) in the large intestine. In the third approach, the porcine IAP isoform-X1 (IAPX1) gene was overexpressed in the prokaryotic ClearColiBL21 (DE3) cell and the recombinant porcine IAPX1 was associated with reduced (p < 0.05) enzyme affinity and maximal enzyme activity. Therefore, levels of glycosylation can modulate plasticity of weaned porcine gut AP functionality towards maintaining gut microbiome and the whole-body physiological status.

Cutting Edge: High-Dose Live Attenuated Influenza Vaccines Elicit Pulmonary Tissue-Resident Memory CD8+ T Cells in the Face of Pre-Existing Humoral Immunity.

In this study, we investigated how pre-existing Ab immunity to influenza virus established from prior immunizations affects the development of CD8+ T cell responses evoked after vaccination with a live attenuated vaccine. Using a mouse model and a panel of live attenuated influenza virus vaccine candidates (cold adapted and single cycle), we show that pre-existing influenza-specific Abs directed against the vaccine backbone attenuate the size and quality of the vaccine-induced CD8+ T cell response. Importantly, we show that increasing the vaccine dose can overcome this impediment, resulting in improved vaccine-induced circulating and tissue-resident memory CD8+ T cell responses, which were protective against heterologous influenza challenge. Thus, the reduced size and quality of the T cell response elicited by a live attenuated influenza virus vaccine imparted by the influenza-specific Ab landscape of the vaccinee can be overcome by increasing vaccine dose.

Characterization of in vitro stability for two processive endoglucanases as exogenous fibre biocatalysts in pig nutrition.

Development of highly efficacious exogenous fibre degradation enzymes can enhance efficiency of dietary fibre utilization and sustainability of global pork production. The objectives of this study were to investigate in vitro stability for two processive endoglucanases, referred to as GH5-tCel5A1 and GH5-p4818Cel5_2A that were overexpressed in CLEARCOLIBL21(DE3). Three-dimensional models predicted presence of Cys residues on the catalytic site surfaces of GH5-tCel5A1 and GH5-p4818Cel5_2A; and time course experimental results shown that both cellulases were susceptible to auto-oxidation by airborne O2 and were unstable. Furthermore, we examined these endoglucanases' stability under the mimicked in vitro porcine gastric and the small intestinal pH and proteases' conditions. Eadie-Hofstee inhibition kinetic analyses showed that GH5-tCel5A1 and GH5-p4818Cel5_2A respectively lost 18 and 68% of their initial activities after 2-h incubations under the gastric conditions and then lost more than 90% of their initial activities after 2-3 h of incubations under the small intestinal conditions. Therefore, further enzyme protein engineering to improve resistance and alternatively post-fermentation enzyme processing such as coating to bypass the gastric-small intestinal environment will be required to enable these two processive endoglucanases as efficacious exogenous fibre enzymes in pig nutrition application.

Tuftelin 1 Facilitates Hepatocellular Carcinoma Progression through Regulation of Lipogenesis and Focal Adhesion Maturation.

Hepatocellular carcinoma (HCC) is the most common type of primary liver malignancy with poor prognosis worldwide. Emerging evidences demonstrated critical roles of lipid de novo synthesis in HCC progression, yet its regulatory mechanisms are not fully understood. Herein, we found that tuftelin 1 (TUFT1), an acidic phosphorylated glycoprotein with secretory capacity, was significantly upregulated in HCC and had an excellent correlation with patient survival and malignancy features. Through database mining and experimental validation, we found that TUFT1 was associated with fatty acid metabolism and promoted lipid accumulation in HCC cells. Further, we found that TUFT1 can interact with CREB1, a transcription factor for hepatic lipid metabolism, and regulate its activity and the transcriptions of key enzymes for lipogenesis. TUFT1 promoted HCC cell proliferation significantly, which was partially reversed by treatment of an inhibitor of CREB1, KG-501. Moreover, TUFT1 promoted the capacity of HCC cell invasion in vitro, which was likely mediated by its association with zyxin, a zinc-binding phosphoprotein responsible for the formation of fully mature focal adhesions on extracellular matrix. We found that TUFT1 can interact with ZYX and inhibit its expression and recruitments to focal complexes in HCC cells. Collectively, our study uncovered new regulatory mechanisms of TUFT1-mediated lipogenesis, cell proliferation, and invasion.

Tissue resident memory T cells in the respiratory tract.

Owing to their capacity to rapidly spread across the population, airborne pathogens represent a significant risk to global health. Indeed, several of the past major global pandemics have been instigated by respiratory pathogens. A greater understanding of the immune cells tasked with protecting the airways from infection will allow for the development of strategies that curb the spread and impact of these airborne diseases. A specific subset of memory T-cell resident in both the upper and lower respiratory tract, termed tissue-resident memory (Trm), have been shown to play an instrumental role in local immune responses against a wide breadth of both viral and bacterial infections. In this review, we discuss factors that influence respiratory tract Trm development, longevity, and immune surveillance and explore vaccination regimes that harness these cells, such approaches represent exciting new strategies that may be utilized to tackle the next global pandemic.

PF-07059013: A Noncovalent Modulator of Hemoglobin for Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu → Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

Airway Exosomes Released During Influenza Virus Infection Serve as a Key Component of the Antiviral Innate Immune Response.

Exosomes are extracellular vesicles secreted by cells that have an important biological function in intercellular communication by transferring biologically active proteins, lipids, and RNAs to neighboring or distant cells. While a role for exosomes in antimicrobial defense has recently emerged, currently very little is known regarding the nature and functional relevance of exosomes generated in vivo, particularly during an active viral infection. Here, we characterized exosomes released into the airways during influenza virus infection. We show that these vesicles dynamically change in protein composition over the course of infection, increasing expression of host proteins with known anti-influenza activity, and viral proteins with the potential to trigger host immune responses. We show that exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigen that can be utilized by antigen presenting cells to drive the induction of a cellular immune response. Moreover, we show that attachment factors for influenza virus, namely α2,3 and α2,6-linked sialic acids, are present on the surface of airway exosomes and these vesicles have the ability to neutralize influenza virus, thereby preventing the virus from binding and entering target cells. These data reveal a novel role for airway exosomes in the antiviral innate immune defense against influenza virus infection.

The long non-coding RNA PVT1/miR-145-5p/ITGB8 axis regulates cell proliferation, apoptosis, migration and invasion in non-small cell lung cancer cells.

Lung cancer is one of the leading causes of death worldwide and non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer. Long noncoding RNAs (lncRNAs) are closely associated with the development and progression of various cancers, including lung cancer. The purpose of this study was to explore the potential role and molecular mechanism of lncRNA plasmacytoma variant translocation 1 (PVT1) in regulating the proliferation, apoptosis, migration, and invasion of NSCLC cells. The expressions of PVT1, integrin ß-8 (ITGB8), and miR-145-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of ITGB8, MEK, p-MEK, ERK, and p-ERK were measured by western blot analysis. Cell proliferation, apoptosis, migration, and invasion were determined by MTT assay, flow cytometry, and transwell assay, respectively. The potential binding sites between miR-145-5p and PVT1 or ITGB8 were predicted by online software and verified by luciferase reporter assay. A xenograft tumor model was established to confirm the effect of PVT1 on NSCLC in vivo. We found out that the expression levels of PVT1 and ITGB8 were upregulated in NSCLC tissues and cells. Knockdown of PVT1 or ITGB8 suppressed cell proliferation, migration, invasion and promoted apoptosis in NSCLC cells, which could be reversed by ITGB8 overexpression in NSCLC cells. Moreover, PVT1 could regulate ITGB8 expression via direct binding to miR-145-5p. Furthermore, PVT1 regulated the MEK/ERK pathway by affecting ITGB8 expression. In addition, knockdown of PVT1 inhibited tumor growth, ITGB8 expression, MEK/ERK signaling pathway, and increased miR-145-5p expression in vivo. In conclusion, the knockdown of PVT1 inhibited proliferation, migration, and invasion but induced apoptosis of NSCLC cells by regulating miR-145-5p/ITGB8 axis and inhibiting MEK/ERK signaling pathway, providing a novel avenue for the treatment of NSCLC.

A processive endoglucanase with multi-substrate specificity is characterized from porcine gut microbiota.

Cellulases play important roles in the dietary fibre digestion in pigs, and have multiple industrial applications. The porcine intestinal microbiota display a unique feature in rapid cellulose digestion. Herein, we have expressed a cellulase gene, p4818Cel5_2A, which singly encoded a catalytic domain belonging to glycoside hydrolase family 5 subfamily 2, and was previously identified from a metagenomic expression library constructed from porcine gut microbiome after feeding grower pigs with a cellulose-supplemented diet. The activity of purified p4818Cel5_2A was maximal at pH 6.0 and 50 °C and displayed resistance to trypsin digestion. This enzyme exhibited activities towards a wide variety of plant polysaccharides, including cellulosic substrates of avicel and solka-Floc®, and the hemicelluloses of ß-(1 → 4)/(1 → 3)-glucans, xyloglucan, glucomannan and galactomannan. Viscosity, reducing sugar distribution and hydrolysis product analyses further revealed that this enzyme was a processive endo-ß-(1 → 4)-glucanase capable of hydrolyzing cellulose into cellobiose and cellotriose as the primary end products. These catalytic features of p4818Cel5_2A were further explored in the context of a three-dimensional homology model. Altogether, results of this study report a microbial processive endoglucanase identified from the porcine gut microbiome, and it may be tailored as an efficient biocatalyst candidate for potential industrial applications.

Apparent and true digestibility of macro and micro nutrients in adult maintenance dog foods containing either a majority of animal or vegetable proteins1.

There is dearth of knowledge with regards to mineral digestibility of ingredients in canines, and current knowledge is focused on the digestibility of supplemented minerals, not on intrinsic mineral digestibility of ingredients. The objectives of the present study were to determine the apparent and true digestibility (TD) of macronutrients and micronutrients, and the total tract gastrointestinal endogenous nutrient outputs in canines fed either animal- or vegetable-based adult maintenance diets. Eight purpose bred Beagles (two intact males, six spayed females) of similar age (2.12 ± 0.35 yr, mean ± SD) and weight (9.92 ± 0.73 kg, mean ± SD) were pair housed in kennels but fed individually based on individual maintenance energy requirements. Two basal diets (animal and vegetable protein based) were formulated to meet nutritional requirements of adult canines. Two additional trial diets were created, using the basal diets, by diluting diets by 50% with anhydrous α-d-glucose to attempt to quantify endogenous mineral losses and enable calculation of TD. All diets contained titanium dioxide at 0.3% for calculations of nutrient digestibility. Dogs were provided with deionized water as their only source of water throughout the trial. Dogs in a specific kennel were randomly assigned to an experimental diet for 10 d (experimental period), and fecal samples were collected the last 4 d of each period. All dogs were fed all experimental diets in random order based on a 4 × 4 replicated Latin square design. Dogs fed intact diets had a higher apparent mineral digestibility compared to dogs fed diluted diets (P < 0.05). Apparent phosphorus digestibility was higher for dogs fed the diet 2 compared with the diet 1 (P = 0.01) and the diluted diets (P < 0.001). There was a trend towards a greater TD of Cu for dogs fed the diet 2 compared with the diet 1 (P = 0.08). P, Mg, Zn, and Mn true digestibilities were higher for dogs fed the diet 2 compared with the diet 1 (P < 0.05, P = 0.01, P = 0.02, P = 0.009, respectively). These results suggest that apparent and TD do not result in similar values. Further research should be conducted on TD in canines only if a better model is developed.

Reimplantation of an extruded osteoarticular segment of the femur: Case series and in vitro study in a rat model.

BACKGROUND: The treatment of open femur fractures with reimplantation of large extruded segments remains one of the most difficult clinical management scenarios. The situation is even complicated when the extruded segments contains a large osteoarticular segment and no consensus exist about the efficient sterilization. We successfully managed five cases of open femur fracture by reimplantation of a large osteoarticular segment. While the outcomes were favourable, we performed an in vitro investigation in a rat model to determine whether the bone segment preparation strategy was optimal. MATERIALS AND METHODS: After meticulous debridement and sterilization with povidone-iodine scrub/orthopaedic antibiotic solution, osteoarticular segments of the femur were reimplanted successfully in five patients with Gustilo-Anderson IIIa-IIIb fractures. Furthermore, in vitro study performed to assess the relative efficacy of various methods of sterilization employed osteoarticular segments of rat femurs. After contamination, osteoarticular segments were treated via one of the following protocols: (1) saline rinse; (2) povidone-iodine scrub and saline rinse; (3) povidone-iodine scrub and autoclaving; (4) povidone-iodine scrub and immersion in antibiotic solution; (5) povidone-iodine scrub and immersion in povidone-iodine solution; or (6) povidone-iodine scrub and gamma-irradiation. The osteoarticular segments were then cultured and finally evaluated for infection and morphological changes. RESULTS: At the mean 40 month follow-up, there were no infection in the patients and the fractures achieved completed union. For the basic research, only approaches involving povidone-iodine scrub with autoclaving or antibiotic solution immersion were 100% effective in eliminating bacterial growth. Furthermore, povidone-iodine scrub with antibiotic solution immersion preserved the articular surface morphology. CONCLUSION: Our study suggests that reimplantation of extruded osteoarticular segments of long bone may represent a feasible alternative to amputation. This is the first description of such a technique and its long-term outcomes in the clinical setting, which were corroborated with the outcomes of in vitro investigation in a rat model, concluding that contaminated extruded osteoarticular segments can be adequately sterilized for reimplantation by cleaning with povidone-iodine scrub followed by brief soaking in antibiotic solution. However, it remains unclear whether the antibacterial efficacy of different sterilizations noted in vitro is reflected in vivo, warranting further research.

Nucleus accumbens-associated protein-1 promotes glycolysis and survival of hypoxic tumor cells via the HDAC4-HIF-1α axis.

Nucleus accumbens-associated protein-1 (NAC1), a nuclear factor of the BTB/POZ gene family, has emerging roles in cancer. In this study, we identified the NAC1-HDAC4-HIF-1α axis as an important pathway in regulating glycolysis and hypoxic adaptation in tumor cells. We show that nuclear NAC1 binds to histone deacetylase type 4 (HDAC4), hindering phosphorylation of HDAC4 at Ser246 and preventing its nuclear export that leads to cytoplasmic degradation of the deacetylase. Accumulation of HDAC4 in the nuclei results in an attenuation of HIF-1α acetylation, enhancing the stabilization and transcriptional activity of HIF-1α and strengthening adaptive response of cells to hypoxia. We also show the role of NAC1 in promoting glycolysis in a mouse xenograft model, and demonstrate that knockdown of NAC1 expression can reinforce the antitumor efficacy of bevacizumab, an inhibitor of angiogenesis. Clinical implication of the NAC1-HDAC4-HIF-1α pathway is suggested by the results showing that expression levels of these proteins are significantly correlative in human tumor specimens and associated with the disease progression. This study not only reveals an important function of NAC1 in regulating glycolysis, but also identifies the NAC1-HDAC4-HIF-1α axis as a novel molecular pathway that promotes survival of hypoxic tumor cells.

Regulation of host epithelial responses to Cryptosporidium infection by microRNAs.

Cryptosporidium species infect the gastrointestinal epithelium and other mucosal surfaces of vertebrate hosts. Epithelial cells provide the first line of defence against Cryptosporidium infection and play a critical role in the initiation, regulation and resolution of both innate and adaptive immune reactions. Host miRNAs in mammalian cells have been shown to play crucial roles in cellular responses to infection by diverse pathogens, including viruses, parasites and bacteria. Given the absence of RNAi machinery in Cryptosporidium, lack of miRNA expression in the parasite and minimal invasion nature of infection, Cryptosporidium infection provides an ideal model for exploring miRNA-mediated epithelial cell defence, relevant to infection of mucosal epithelial cells by pathogens in general. Increasing evidence supports that miRNAs may modulate many stages of epithelial responses following Cryptosporidium infection, including activation of the intracellular signalling pathways, production of antimicrobial molecules, expression of cytokines/chemokines, release of epithelial cell-derived exosomes and feedback regulation of immune homeostasis. On the other hand, this parasite may have developed strategies to modulate host miRNA-mediated cellular function for immune evasion. In this review, we will summarize the recent advances on miRNA regulation of epithelial responses to Cryptosporidium infection, with an emphasis on host defence and parasite immune evasion.

Chemoselective Preparation of Clickable Aryl Sulfonyl Fluoride Monomers: A Toolbox of Highly Functionalized Intermediates for Chemical Biology Probe Synthesis.

Sulfonyl fluoride (SF)-based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF-sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)-mediated azide-alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.