Biochemical Analysis Research Articles

Protective effect and mechanism of Zuogui Jiangtang Jieyu Formula on damage to hippocampal synaptic microenvironment in rats with diabetes-related depression based on microglia-neuron crosstalk signal CD300f/TLR4

This study aims to reveal the protective effect and mechanism of Zuogui Jiangtang Jieyu Formula on the damage to hippo-campal synaptic microenvironment in rats with diabetes-related depression(DD) via regulating microglia immune receptor molecule-like family member f(CD300f)/Toll-like receptor 4(TLR4) signal. Firstly, the model of DD rats was established by a two-week high-fat diet+STZ injection+chronic mild and unpredictable stress plus isolation for 28 days. The rats were randomly divided into normal group, model group, CD300f blocker(CLM1, 2 μg·kg~(-1)) group, CD300f agonist(Fcγ, 5 μg·kg~(-1)) group, positive drug(0.18 g·kg~(-1) metformin+1.8 mg·kg~(-1) fluoxetine) group, and high-dose and low-dose(20.52 and 10.26 g·kg~(-1)) Zuogui Jiangtang Jieyu Formula groups. Depression-like behavior of rats was evaluated by open field and forced swimming experiments. The levels of blood glucose and insulin were detected by biochemical analysis. The levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β), indoleamine 2, 3-dioxygenase(IDO), 5-hydroxytryptamine(5-HT), and dopamine(DA) in the hippocampus were detected by enzyme-linked immunosorbent assay. The changes in the synaptic ultrastructure in hippocampal neurons of rats were observed by transmission electron microscopy. The protein expressions of CD300f, TLR4, synaptophysin(SYN), and postsynaptic density protein 95(PSD-95) in microglial cells of the hippocampus were detected by immunofluorescence and Western blot. The results indicated that compared with that in the normal group, the total movement distance in open field experiments was reduced in the model group, and the immobility time in forced swimming experiments increased, with an elevated insulin level in serum, as well as TNF-α, IL-1β, and IDO levels in the hippocampus. The 5-HT and DA levels in the hippocampus were reduced. In addition, the CD300f expression was down-regulated in microglial cells of the hippocampus, and the TLR4 expression was up-regulated. Moreover, the expression of synapse-related proteins SYN and PSD-95 in hippocampal neurons decreased, and the synaptic ultrastructure of hippocampal neurons was significantly damaged. Compared with the model group, the CD300f blocker and agonist aggravated and alleviated the above abnormal changes, respectively. High-dose and low-dose Zuogui Jiangtang Jieyu Formula could significantly improve the above depression-like beha-vior in rats, inhibit the abnormal increase of TNF-α, IL-1β, and IDO and the decrease of 5-HT and DA, effectively increase the expression of CD300f in microglial cells, and decrease the expression of TLR4. They could up-regulate the protein expression of presyna-ptic membrane SYN and postsynaptic membrane PSD-95 in hippocampal neurons and finally improve the damage to the hippocampal synaptic microenvironment. In conclusion, this research confirmed that Zuogui Jiangtang Jieyu Formula effectively alleviated the depression-like behavior and inhibited inflammatory activation of microglial cells in the hippocampus of rats with DD, and the mechanism might be related to the regulation of CD300f/TLR4 signal to alleviate the damage to hippocampal synaptic microenvironment.

Investigation of Urotensin II expression in placenta and umbilical cord in pregnancies with intrauterine growth restriction by histological and biochemical methods

ObjectiveIn this study, it was aimed to investigate Urotensin II in intrauterine growth restriction (IUGR) and its connection to autophagy and/or apoptosis in placenta and umbilical cord by immunohistochemical and biochemical methods. Materials and methodsThe study included 30 healthy pregnant women and 30 pregnant women with IUGR, aged 19–45, at Atatürk University Gynecology Clinic. Samples were collected from placenta, umbilical cord, maternal blood, and umbilical cord blood during delivery. Histopathological examination was carried out on placenta and umbilical cord, and UTII, Beclin 1, and caspase 3 expressions were analyzed in these tissues. Biochemical analysis was performed on maternal and umbilical cord serum samples. ResultsIn healthy placentas, normal villus formation was seen, but those with IUGR showed accelerated villus maturation, causing inadequate nutrition and development. IUGR placentas had fibrin deposition, villous edema, syncytial nodes increase, and intervillous distance. Umbilical cords of IUGR group had differences in vessel wall thickness, arterial lumens, and vessel number. Higher levels of UTII, Beclin 1, and caspase 3 were found in IUGR placenta and cord. Beclin 1 and caspase 3 levels were significantly higher in IUGR group compared to controls, while UTII levels were not significantly different in maternal and cord serums. ConclusionAs a result of our findings, UTII increase in placenta and umbilical cord may lead to IUGR formation by inducing autophagy and apoptosis.

O10 HEART RATE VARIABILITY AS EARLY MARKER OF CARDIAC INJURY IN HYPERTENSIVE YOUNG ADULTS

Background: Hypertension remains a life-threatening risk factor for cardiovascular diseases globally. It is not only common to middle age or aged group but also affect young individuals. Heart rate variability (HRV) gives an inference of the overall cardiac function and health of an individual. Thus, the aim of this study was to investigate whether HRV could be an early marker and a valid alternative to biochemical probes for the early detection of cardiac injury and systemic inflammation in hypertensive young adults. Materials and methods: A total number of 300 participants were recruited by fixed sampling of the case control study grouped into normotensive and hypertensive young groups. Informed consent was obtained from all participants before the procedure. Blood pressure with some anthropometric data were collected. Afterward, each participant’s HRV data was acquired by conducting a 5-minute resting ECG. Then, 5 ml of blood was taken through sterile venepuncture, centrifuged at 3000 rpm for 15 mins and biochemical analysis was performed for plasma troponin-I, troponin-T and inflammatory markers (TNF-alpha and NF-kappaB). Results: HRV assessment showed significant decrease in HRV parameters of parasympathetic activity (SDNN, RMSSD, HF) in the hypertensive young group. LF also showed significant decrease in the hypertensive group. But, LF/HF (sympatho-vagal balance) showed significant increase in the hypertensive group. LF correlates with plasma TNF-α (r = -0.6167) and NF-kB (r = -0.6673) at p = 0.0003 and p<0.0001, respectively. In addition, LF correlates with plasma Troponin-I (r = -0.6393) and Troponin-T (r = -0.5719) at p = 0.0001 and p = 0.0010, respectively. LF/HF had no correlation with these biomarkers. Thus, HRV correlates with plasma troponin-I, troponin-T, TNF-alpha and NF-kappaB in hypertensive young adults. Conclusion: HRV can be used as an early marker of cardiac injury and inflammation in young hypertensive individuals.

Arsenic Induced Oxidative Neural-Damages in Rat are Mitigated by Tea-Leave Extract via MMPs and AChE Inactivation, Shown by Molecular Docking and in Vitro Studies with Pure Theaflavin and AChE.

Chronic arsenic-exposure causes neuromuscular disorders and other health anomalies. Damage to DNA and cytoskeletal/extracellular matrix is brought on by reactive-oxygen-species (ROS)-induced intrinsic antioxidant depletion (thiols/urate). Therapeutic chelating-agents have multiple side-effects. The protection of (Camellia sinensis) tea-extract and role of uric-acid (UA) or allopurinol (urate-depletor) on arsenic-toxicity were verified in rat model. Camellia sinensis (CS dry-leaves), UA or allopurinol was supplemented to arsenic-intoxicated rats for 4-weeks. Purified theaflavins and their galloyl-ester were tested in-vitro on pure AChE (acetylcholinesterase) and their PDB/PubChem 3-D structures were utilized for in-silico binding studies. The primary chemical components were evaluated from CS-extracts. Biochemical analysis, PAGE-zymogram, DNA-stability comet analysis, HE-staining was performed in arsenic-exposed rat brain tissues. Animals exposed to arsenic showed symptoms of erratic locomotion, decreased intrinsic antioxidants (catalase/SOD1/uric acid), increased AChE, and malondialdehyde. Cerebellar and cerebrum tissue damages were shown with increased levels of matrix-metalloprotease (MMP2/9) and DNA damage (comets). Allopurinol- supplemented group demonstrated somewhat similar biochemical responses. In the CS-group brain tissues especially cerebellum is considerably protected which is evident from endogenous antioxidant and DNA and cytoskeleton protection with concomitant inactivation of MMPs and AChE. Present study indicates theaflavin-digallate (TFDG) demonstrated the highest inhibition of purified AChE (IC50 = 2.19 µg/ml with the lowest binding free-energy; -369.87 kcal/mol) followed by TFMG (IC50 = 3.86 µg/ml, -347.06 kcal/mol) suggesting their possible restoring effects of cholinergic response. Favorable responses in UA-group and adverse outcome in allo-group justify the neuro-protective effects of UA as an endogenous antioxidant. Role of flavon-gallate in neuro protection mechanism may be further studied.

Biochemical approaches for decoding the information stored with metabolites

Molecular data storage is an emerging supplement to conventional information storage methods. In addition to the information storage with DNA or synthetic polymers, a recently proposed strategy that uses small molecule mixtures as information carriers has sparked interest since it is a synthesis-free method. Here, we demonstrate two methods for decoding digital information stored in two sets of small-molecule metabolites. The information is stored as binary code, 0 or 1, based on the absence or presence of each particular metabolite in the mixture. In the first approach, the information was encoded by five metabolites (sarcosine, cholesterol, xanthine, glucose, and galactose) and stored as mixture solutions in a 384-well microplate. The information was read out by cascade enzymatic reactions that can specifically convert the metabolites to a colorimetric product, which was then assayed by a microplate reader. We stored a 60×32-pixel picture (1920 bits) with 384 mixtures of small molecules with a reading accuracy of 96.68 %, and also demonstrated the encoding of a 220-bit text message. Alternatively, another set of six metabolites (bilirubin, uric acid, urea, creatinine, glucose, and triglyceride) was deployed to encode a 78×102-pixel picture (7956 bits) with 1326 mixtures, which was decoded automatically by a biochemistry analyzer with a 98.96 % accuracy. This work showcases that common metabolites can be used as digital molecules for information storage, and the information decoding can be done by accessible instruments in an ordinary biochemical laboratory.

Biochemical profiling, organoleptic assessment, and functional property analysis of the health supplement

Biochemical profiling, organoleptic assessment, and functional property analysis of the health supplement

Efficient parameter estimation in biochemical pathways: Overcoming data limitations with constrained regularization and fuzzy inference

In analytical modeling for biochemical pathways, precisely determining unknown parameters is paramount. Traditional methods, reliant on experimental time course data, often encounter roadblocks — limited accessibility and variable quality — that can significantly impact the algorithm’s performance. In this study, we address these hurdles by unveiling a groundbreaking parameter estimation technique, Constrained Regularized Fuzzy Inferred Extended Kalman Filter (CRFIEKF). This innovative approach eliminates the need for experimental time-course measurements and capitalizes on the existing imprecise relationships among the molecules within the network. Our proposed framework integrates a Fuzzy Inference System (FIS) block to encapsulate these approximated relationships. To fine-tune the estimated parameter values, we employ Tikhonov regularization. The selection of Tikhonov regularization and Gaussian membership functions was based on the Mean Squared Error (MSE) values observed during the parameter estimation process, contrasting our results with those of previous studies.We rigorously tested the proposed approach across various pathways, from the glycolytic processes in mammalian erythrocytes and yeast cells to the intricate JAK/STAT and Ras signaling pathways. The results were impressive, showing a significant similarity (p-value < 0.001) to the outcomes of specific prior experiments. The dynamics of the biochemical networks normalized within the [0, 1] range mirrored the transient behavior (MSE < 0.5) of both in vivo and in silico results from previous studies. In conclusion, our findings highlight the effectiveness of CRFIEKF in estimating the kinetic parameter values without prior knowledge of experimental data within a biochemical pathway in the state-space model. The proposed method underscores its potential as a game-changer in biochemical pathway analysis.

Trimetazidine Alleviates Bleomycin-Induced Pulmonary Fibrosis by Targeting the Long Noncoding RNA CBR3-AS1-Mediated miRNA-29 and Resistin-Like Molecule alpha 1: Deciphering a Novel Trifecta Role of LncRNA CBR3-AS1/miRNA-29/FIZZ1 Axis in Lung Fibrosis.

Pulmonary fibrosis (PF) and tissue remodeling can greatly impair pulmonary function and often lead to fatal outcomes. In the present study, we explored a novel molecular interplay of long noncoding (Lnc) RNA CBR3-AS1/ miRNA-29/ FIZZ1 axis in moderating the inflammatory processes, immunological responses, and oxidative stress pathways in bleomycin (BLM)-induced lung fibrosis. Furthermore, we investigated the pharmacological potential of Trimetazidine (TMZ) in ameliorating lung fibrosis. Our results revealed that the BLM-treated group exhibited a significant upregulation in the expression of epigenetic regulators, lncRNA CBR3-AS1 and FIZZ1, compared to the control group (P<0.0001), along with the downregulation of miRNA-29 expression. Furthermore, Correlation analysis showed a significant positive association between lnc CBR3-AS1 and FIZZ1 (R=0.7723, p<0.05) and a significant negative association between miRNA-29 and FIZZ1 (R=-0.7535, p<0.05), suggesting lnc CBR3-AS1 as an epigenetic regulator of FIZZ1 in lung fibrosis. BLM treatment significantly increased the expression of Notch, Jagged1, Smad3, TGFB1, and hydroxyproline. Interestingly, the administration of TMZ demonstrated the ability to attenuate the deterioration effects caused by BLM treatment, as indicated by biochemical and histological analyses. Our investigations revealed that the therapeutic potential of TMZ as an antifibrotic drug could be ascribed to its ability to directly target the epigenetic regulators lncRNA CBR3-AS1/ miRNA-29/ FIZZ1, which in turn resulted in the mitigation of lung fibrosis. Histological and immunohistochemical analyses further validated the potential antifibrotic effects of TMZ by mitigating the structural damage associated with fibrosis. Taken together, our study showed for the first time the interplay between epigenetic lncRNAs CBR3-AS1 and miRNA-29 in lung fibrosis and demonstrated that FIZZ1 could be a downregulatory gene for lncRNA CBR3-AS1 and miRNA-29. Our key findings demonstrate that TMZ significantly reduces the expression of fibrotic, oxidative stress, immunomodulatory, and inflammatory markers, along with epigenetic regulators associated with lung fibrosis. This validates its potential as an effective antifibrotic agent by targeting the CBR3-AS1/miRNA-29/FIZZ1 axis.

Network Pharmacology and Experimental Analysis to Explore the Effect and Mechanism of Modified Buyang Huanwu Decoction in the Treatment of Diabetic Nephropathy.

Preventing and treating diabetic nephropathy (DN) are global challenges due to the complexity and diversity of its causes and manifestations. It is important to find effective medications to treat DN. Gene expression files of DN were downloaded from the GEO database to identify the differentially expressed genes. Network pharmacology and molecular docking were used to explore the possible mechanisms of modified Buyang Huanwu Decoction (mBHD) in treating DN. Biochemical, histopathological, and real-time PCR analyses were conducted in both in vivo and in vitro DN models to investigate the effects of mBHD. A total of 336 active ingredients and 124 potential targets of mBHD associated with DN were identified. Among them, 8 hub genes were found to be important targets for mBHD in treating DN and were significantly correlated with the infiltration status of six immune cells. Partially, the active ingredients of mBHD demonstrated good stability in binding to CASP3 and TP53. mBHD treatment significantly reduced levels of total cholesterol, triglyceride, blood urea nitrogen, serum creatinine, and microalbumin in db/db mice. HE and Masson's staining results showed that mBHD attenuated renal injury in db/db mice. Additionally, mBHD treatment could significantly alter the expression of CASP3, CCL2, TP53, ALB, and HMOX1. mBHD may be involved in the treatment of DN through multiple ingredients, targets, and pathways. In addition, mBHD could alleviate renal injury in db/db mice, possibly involving CASP3, CCL2, TP53, ALB, and HMOX1.

Saroglitazar, a PPAR α/γ agonist alleviates 3-Nitropropionic acid induced neurotoxicity in rats: Unveiling the underlying mechanisms

Saroglitazar (SGZ), a peroxisomal proliferated activated receptor α/γ agonist showed neuroprotective effects in various neurodegenerative disorders like Alzheimer’s and Parkinson’s. However, no studies were performed on Huntington’s, so the goal of the current study is to examine the effect of SGZ on Huntington’s disease like symptoms induced by 3-Nitropropionic acid. In this protocol, twenty-four rats were divided into four groups, each group consisting of 6 animals. Group 1: The control group received 1% CMC 10mg/kg, p.o. for 14 days. Groups 2, 3, and 4 received 3-NP 15mg/kg, i.p. from Day 1 to Day 7. Groups 3 and 4 received SGZ 5mg/kg, p.o. and 10mg/kg, p.o. respectively once daily from day 1 to day 14. Various behavioral tests like OFT, rotarod, hanging wire, narrow beam walk, MWM, and Y-maze were performed. On day-15, the animals were euthanised by cervical dislocation and brain sample were isolated for biochemical and histopathological analysis. Administration of 3-NP showed a significant decrease in motor coordination and cognitive function. Furthermore, 3-NP altered the activity of acetylcholinesterase, anti-oxidant enzymes, Nrf-2, NF-κB, BDNF, CREB levels, and histological features. However, treatment with SGZ showed ameliorative effects in the 3-NP induced neurotoxicity via PPAR α/γ pathway by reducing motor dysfunction, memory impairment, cholinesterase levels, oxidative stress, neuroinflammation. It also enhanced the levels of Nrf-2, BDNF, and CREB expression and improved histological features. In conclusion, treatment with Saroglitazar attenuated Huntington’s disease-like symptoms in rats which are induced by 3-NP via activation of PPAR α/γ pathway.

Twenty-eight days of repeated dose sub-acute toxicological evaluation of polyherbal Ayurvedic medicine BPGrit in Sprague-Dawley rats.

A pre-clinical toxicological evaluation of herbal medicines is necessary to identify any underlying health-associated side effects, if any. BPGrit is an Ayurveda-based medicine prescribed for treating hypertensive conditions. High-performance liquid chromatography-based analysis revealed the presence of gallic acid, ellagic acid, coumarin, cinnamic acid, guggulsterone E, and guggulsterone Z in BPGrit. For sub-acute toxicity analysis of BPGrit, male and female Sprague-Dawley rats were given repeated oral gavage at 100, 300, and 1000 mg/kg body weight/day dosages for 28 days, followed by a 14-day recovery phase. No incidences of mortality, morbidity, or abnormal clinical signs were observed in BPGrit-treated rats throughout the study period. Also, the body weight and food consumption habits of the experimental animals did not change during the study duration. Hematological, biochemical, and histopathological analysis did not indicate any abnormal changes occurring in the BPGrit-treated rats up to the highest tested dose of 1000 mg/kg body weight/day. Finally, the study established the "no-observed-adverse-effect level" for BPGrit at >1000 mg/kg body weight/day in Sprague-Dawley rats.

Structural basis of the human transcriptional Mediator regulated by its dissociable kinase module.

The eukaryotic transcriptional Mediator comprises a large core (cMED) and a dissociable CDK8 kinase module (CKM). cMED recruits RNA polymerase II (RNA Pol II) and promotes pre-initiation complex formation in a manner repressed by the CKM through mechanisms presently unknown. Herein, we report cryoelectron microscopy structures of the complete human Mediator and its CKM. The CKM binds to multiple regions on cMED through both MED12 and MED13, including a large intrinsically disordered region (IDR) in the latter. MED12 and MED13 together anchor the CKM to the cMED hook, positioning CDK8 downstream and proximal to the transcription start site. Notably, the MED13 IDR obstructs the recruitment of RNA Pol II/MED26 onto cMED by direct occlusion of their respective binding sites, leading to functional repression of cMED-dependent transcription. Combined with biochemical and functional analyses, these structures provide a conserved mechanistic framework to explain the basis for CKM-mediated repression of cMED function.

Safety evaluation of Avicenna germinans leaf extracts in rats

BackgroundAvicenna germinans is one of Nigeria's dominant mangrove plants and has valuable pharmacological and therapeutic activity. It is widely employed in traditional medicine to treat many ailments and diseases, including cancer. However, little is known about the safety of the plant. Acute and sub-acute toxicity profiles of the plant extracts were assessed in this study. MethodsThe oral acute toxicity was determined with 5000 mg/kg body weight each of n-hexane, ethyl acetate, and ethanol extracts of Avicenna germinans leaf in rats. The rats were monitored for mortality, clinical manifestations, and weight changes over 14 days. During the sub-acute toxicity study, twenty-five female rats were fed with 0–1000 mg/kg ethanol extracts of Avicenna germinans for 21 days. The body weight was monitored during treatment, while changes in gross organ morphology, relative organ weights, hematology, serum biochemistry, and limited histological analysis were evaluated after treatment. ResultsAcute administration of the three extracts did not result in mortality or any obvious adverse effects. The gross pathology, relative organ weights, hematology, serum biochemistry, and histopathology were similar to the control, except for the 1000 mg/kg dose that evoked marked increases in relative heart, intestinal, thymus, and adrenal weights. In addition, foci of inflammatory cells in the interstitium and lymphoid hyperplasia were found in the heart and lungs, respectively, in the 1000 mg/kg group. ConclusionThe findings indicate that LD50 of the three extracts of Avicenna germinans were > 5000 mg/kg and sub-acute administration of the ethanol extract is relatively safe at least up to 750 mg/kg body weights.

Biochemical Defense Arsenal, Genes/QTLs and Transcripts for Imparting Anthracnose Resistance in Common bean (Phaseolus vulgaris L.)

Anthracnose (ANT), caused by Colletotrichum lindemuthianum, is the most devastating disease affecting common bean (Phaseolus vulgaris L.), leading to significant yield losses in the Western Himalayas. The study provides a comprehensive understanding of ANT resistance via trait phenotyping, biochemical profiling, genome-wide association studies (GWASs), and RNA sequencing. The assessment of bean association mapping panel in different environments revealed a diverse spectrum of resistance levels. Biochemical analysis revealed distinctive defense responses against ANT infection among different genotypes. GWAS approach identified 24 significant marker‒trait associations (MTAs) distributed across all 11 bean chromosomes. Notably, 03 MTAs (BMr205, BMr269 and BMr244) present on chromosome Pv07 were validated for ANT, and the remaining MTAs were novel MTAs for ANT. Transcriptome sequencing of resistant (PBG-3) and susceptible (PBG-26) genotypes under mock and 120-hour post inoculation conditions revealed key differentially expressed genes, such as leucine-rich repeat domain-containing protein (PHAVU_007G087700g), NB-ARC domain-containing protein (PHAVU_003G002500g) and transcription factors pivotal for disease resistance. The expression patterns of four genes (PHAVU_007G087700g, PHAVU_003G002500g, PHAVU_007G056100g and PHAVU_003G003000g) were validated through quantitative reverse transcription polymerase chain reaction (qRT‒PCR). Furthermore, the integration of GWAS-identified candidate genes with transcriptomics and cross-referencing with previous studies validated overlapping regions and common candidate genes, enriching our understanding of the genetic basis of ANT resistance. Therefore, the results offer a holistic perspective on ANT resistance in common bean, providing a foundation for targeted breeding efforts. The identified potential candidate genes and associated pathways will contribute valuable insights into the development of ANT resistant common bean varieties.

Structural and biochemical characterization of the C-terminal region of the human RTEL1 helicase.

RTEL1 is an essential DNA helicase which plays an important role in various aspects of genome stability, from telomere metabolism to DNA replication, repair and recombination. RTEL1 has been implicated in a number of genetic diseases and cancer development, including glioma, breast, lung and gastrointestinal tumors. RTEL1 is a FeS helicase but, in addition to the helicase core, it comprises a long C-terminal region which includes a number of folded domains connected by intrinsically disordered loops and mediates RTEL1 interaction with factors involved in pivotal cellular pathways. However, information on the architecture and the function of this region is still limited. We expressed and purified a variety of fragments encompassing the folded domains and the unstructured regions. We determined the crystal structure of the second repeat, confirming that it has a fold similar to the harmonin homology domains. SAXS data provide low-resolution information on all the fragments and suggest that the presence of the RING domain affects the overall architecture of the C-terminal region, making the structure significantly more compact. NMR data provide experimental information on the interaction between PCNA and the RTEL1 C-terminal region, revealing a putative low-affinity additional site of interaction. A biochemical analysis shows that the C-terminal region, in addition to a preference for telomeric RNA and DNA G-quadruplexes, has a high affinity for R-loops and D-loops, consistent with the role played by the RTEL1 helicase in homologous recombination, telomere maintenance and preventing replication-transcription conflicts. We further dissected the contribution of each domain in binding different substrates.

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Comparative biochemical analysis of platelet-rich plasma-chitosan and platelet-rich fibrin-chitosan for treating tibial bone defects in rabbits

Comparative biochemical analysis of platelet-rich plasma-chitosan and platelet-rich fibrin-chitosan for treating tibial bone defects in rabbits

Quasi-solid electrochemiluminescence immunosensor constructed with gel electrolyte for the detection of CEA

In this paper, we report a novel quasi-solid electrochemiluminescence (ECL) immunosensor platform for the detection of tumor marker carcinoembryonic antigen (CEA). Referring to the conventional three-electrode system of electrochemistry, a gel alternative solution was introduced, and a quasi-solid solvent-free scheme was designed. The use of gels brings several advantages. Firstly, the introduction of gels also provides a new platform for the specific recognition of antigens and antibodies. Secondly, gels can be mass-manufactured like ELISA plate, which reduces the cost of testing and thus provides a reference scheme for large-scale sensor applications. Thirdly, a new device was designed to accommodate the improved sensors. In addition, a simple one-step hydrothermal method was used to synthesize AuNPs/MnO2/TiO2 materials. Utilizing its excellent catalytic activity for CO-reaction reagents H₂C₂O₄, it promotes the production of CO2–• and thus enhances the ECL of Ru(bpy)32+. Thus, this biosensor provides a novel and efficient strategy for CEA detection. The gel system sensor also provides a general model for the detection of other biomarkers and provides new applications for biochemical analysis and clinical diagnosis.

Structural basis for substrate flexibility of the O-methyltransferase MpaG' involved in mycophenolic acid biosynthesis.

MpaG' is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase involved in the compartmentalized biosynthesis of mycophenolic acid (MPA), a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. MpaG' catalyzes the 5-O-methylation of three precursors in MPA biosynthesis including demethylmycophenolic acid (DMMPA), 4-farnesyl-3,5-dihydroxy-6-methylphthalide (FDHMP), and an intermediate containing three fewer carbon atoms compared to FDHMP (FDHMP-3C) with different catalytic efficiencies. Here, we report the crystal structures of S-adenosyl-L-homocysteine (SAH)/DMMPA-bound MpaG', SAH/FDHMP-3C-bound MpaG', and SAH/FDHMP-bound MpaG' to understand the catalytic mechanism of MpaG'and structural basis for its substrate flexibility. Structural and biochemical analyses reveal that MpaG' utilizes the catalytic dyad H306-E362 to deprotonate the C5 hydroxyl group of the substrates for the following methylation. The three substrates with differently modified farnesyl moieties are well accommodated in a large semi-open substrate binding pocket with the orientation of their phthalide moiety almost identical. Based on the structure-directed mutagenesis, a single mutant MpaG'Q267A is engineered with significantly improved catalytic efficiency for all three substrates. This study expands the mechanistic understanding and the pocket engineering strategy for O-methyltransferases involved in fungal natural product biosynthesis. Our research also highlights the potential of O-methyltransferases to modify diverse substrates by protein design and engineering.

Catalpol ameliorates liver fibrosis via inhibiting aerobic glycolysis by EphA2/FAK/Src signaling pathway

BackgroundHepatic fibrosis is a pathological process in a variety of acute or chronic liver injuries. Catalpol (CAT), an iridoid glycoside found in Rehmannia glutinosa, has several pharmacological properties, including anti-inflammatory, antidiabetic and anti-fibrotic effects. Nevertheless, there is currently no report on whether CAT regulates the aerobic glycolysis of hepatic stellate cells (HSCs) to inhibit liver fibrosis. ObjectiveThis study aimed to investigate the protective effects of CAT on hepatic fibrosis and elucidate its underlying mechanisms. MethodsTo explore whether CAT improved liver fibrosis in vivo and in vitro, hepatic fibrosis was induced to mice by intraperitoneally injecting carbon tetrachloride (CCl4). Additionally, LX-2 cells were stimulated with transforming growth factor-β (TGF-β) to simulate fibrosis in vitro. Serum markers of liver injury were examined by using an automatic biochemical analyzer. Histopathological staining, Immunofluorescence (IF) staining, Western blot (WB) analysis, co-immunoprecipitation (Co-IP), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), etc. were employed to identify the targeting between CAT and EphA2 and detect the expression of aerobic glycolysis related proteins, fiber markers and signaling pathways that are responsible for CAT's anti-fibrotic effects of CAT. ResultsResults showed that CAT significantly inhibited hepatic injury, fibrogenesis and inflammation in mice treated with CCl4. This was demonstrated by the enhancement of fibrosis markers, liver function indices, and histopathology. In addition, CAT significantly inhibited the activation of HSCs in TGF-β-induced LX-2 cells, as indicated by decreased proliferation, migration, and expression of collagen I and a-SMA. The study results also suggested that CAT may exert anti-fibrotic effects by inhibiting glycolysis in activated HSCs and in CCl4-treated mice. Mechanistically, CAT directly targets Ephrin type-A receptor 2 (EphA2) to reduce binding with focal adhesion kinases (FAK) and significantly inhibits the FAK/Src pathway. In addition, the pharmacological inhibition of EphA2 cannot further increase the therapeutic effects of CAT on liver fibrosis in vivo and in vitro. ConclusionThe study findings generally demonstrated that CAT presented a novel therapeutic method to treat hepatic fibrosis; this method which inhibits the aerobic glycolysis of activated HSCs through the EphA2/FAK/Src signaling pathway.