Alpinia galanga leaves (ALE), Alpinia galanga rhizomes (ARE), Vitis vinifera seeds (VSE), Moringa oleifera leaves (MLE), Panax ginseng leaves (PLE) and Panax ginseng rhizomes (PRE) ethanolic extracts on human neuroblastoma (SHSY5Y) cells. The 1-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging of VSE and MLE were 81% and 58%, respectively. Ferric-reducing antioxidant power (FRAP) of ALE and MLE (33.57 ± 0.20 and 26.76 ± 0.30 μmol Fe(ΙΙ)/g dry wt., respectively) were higher than for the other extracts. Liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) revealed MLE active compounds. Intracellular study by nitro-blue tetrazolium (NBT) test showed that MLE and VSE had high O2− scavenging . MLE had the highest ROS scavenging followed by PRE , by 2,7-dichlorodihydrofluorescein diacetate (DCFHDA) assay. The 3--2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and neuroprotection tests on SHSY5Y showed that PRE had a better neuroprotective effect but higher cytotoxicity compared to MLE . In conclusion, among the studied plants, MLE has potential for developing as a neuroprotective agent.The current trend worldwide is searching plant extracts towards prevention of neurodegenerative disorders. This study aimed to investigate the neuroprotective effect of  There are many factors that affect ROS production in cells. The key producer of ROS is environmental stress, and ROS are a common byproduct of the normal metabolic pathway of oxygen molecules. Increased ROS levels either outside or inside the cells result in significant damage to all biological macromolecules, namely lipids, proteins, and nucleic acids. Cells naturally generate ROS such as O2− and H2O2 through one-electron reduction of oxygen to O2− catalyzed by an NADPH or NADH oxidase utilizing NADPH or NADH as electron donors. Part of the O2− is converted to H2O2 via spontaneous or enzyme-facilitated dismutase; this reaction is called oxidative burst. The H2O2 causes significant physiological and pathological effects due to its being highly diffusible and its ability to cross the plasma membrane [Reactive oxygen species (ROS) are a group of oxygen-containing molecules that are highly reactive due to the presence of unpaired electrons; ROS mainly include hydroxyl radical (OH•), superoxide anion  due to its requiring a large quantity of oxygen to maintain its normal function. Furthermore, neuronal cells are more sensitive to oxidative stress than the cells in other types of tissue. Therefore, the inhibition of oxidative stress is considered to be a strategy to prevent neuronal progressive disease [The brain is an organ that is prone to producing ROS .Many plants rich in phenolic compounds are consumed almost daily by Asian people in their diet; selecting and investigating the antioxidant effects of the most commonly used plants will provide a source of natural, reliable, cheap, and safe neuroprotection compounds for everyone. The traditional use of ollicles . Alpiniatial oil . In variotstocks . Panax ge system . In thisM. oleifera leaves extract (MLE) related to antioxidant capacity were elucidated by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC–QTOF/MS). This is the first report on the investigation of M. oleifera cultivated in Thailand in both antioxidant actions (capacity and activity) as well as providing profile analysis of valuable phenolic compounds, particularly flavonoids. This information could pave the way for further study of their antioxidant activities and effects as a single compound on neuroprotective agents that exhibit significance for mental health protection, particularly those for neurodegenerative disorders induced by oxidative stress.SHSY5Y cell line is a subline of the SK-N-SH cell line, established in 1970 from a bone marrow biopsy of a neuroblastoma tumor of a four-year-old female in the metastatic stage that underwent three rounds of clonal selection . In addiA. galanga, and P. ginseng and leaves of M. oleifera were obtained from local gardens in Khon Kaen, Thailand in October 2017, whereas V. vinifera seeds were provided by Visootha (Nikki) Lohinavy of GranMonte Asoke Valley Winery from a winemaking factory in Pak Chong, Nakorn Rachasima, Thailand. Plant materials were cleaned, dried at 65 °C using a hot air oven , and crushed using a Philips mill . The ground materials were kept in screwcap containers at room temperature away from the sun until the beginning of the extraction process.Leaves and rhizomes of the selected plant species V. vinifera seeds [M. oleifera leaves [P. ginseng leaves and rhizomes [A. galanga leaves and rhizomes were extracted with 95% ethanol [A. galanga rhizomes extract (ARE) consisted of two layers, with a pale yellow upper layer (PYL) with oily characteristics and a brown lower layer (BL). A. galanga leaves extract (ALE), P. ginseng leaves and rhizomes extracts (PLE and PRE), V. vinifera seeds extract (VSE) and M. oleifera leaves extract (MLE) were obtained in powder form while ARE (both BL and PYL) was in semi-solid form due to the presence of essential oil. Each extract was kept separately in screwcap vials at 4 °C.All plant samples were subjected to extraction using ethanol with a ratio of 1:4 and stirring with a magnetic stirrer for 8 h, then were filtrated using Whatman No. 1 filter paper . ra seeds ,13, M. oa leaves ,15, and rhizomes ,17 were  ethanol ,19. EachAb0 is the absorbance of the control (no extract) and Ab1 is the absorbance of the test extract.The estimation of the free DPPH radical system was performed as described in . The rea3. 6H2O). Distilled water was used as a negative control while Trolox was used as a positive control (with 0.03 ± 0.1 mg/mL for ferric-reducing power). The reaction was started by adding 100 µL of crude extract into 2 mL of the FRAP working solution and kept for 30 min in dark conditions. The absorption of the ferrous tripyridyltriazine complex was recorded at 593 nm. The standard curve was obtained by various concentrations of ferrous sulfate ranging from 5 to 100 µM/mL. The results of FRAP were expressed in μmol Fe (ΙΙ)/g dry weight.The FRAP assay was achieved according to  with som®), CRL2266™, Manassas, VA, USA) was obtained from the Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand. The medium DMEM/F-12 containing Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 Nutrient Mixture at a ratio of 1:1 was used in the SHSY5Y culture. Cells were grown in DMEM/F-12 medium containing 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, 1.5 g/L sodium bicarbonate, 100 U/mL penicillin, and 100 µg/mL streptomycin supplemented with 10% heat-activated fetal bovine serum (FBS)  and incubated at 37 °C in an incubator with 85% humidified atmosphere containing 5% CO2.The SHSY5Y cell line (human neuroblastoma) -2,5-diphenyltetrazolium bromide (MTT) in Dulbecco’s phosphate buffer saline (DPBS) was added, and cells were incubated for an additional 4 h. Finally, MTT solution was gently removed and 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve crystal formazan. Triton X-100 was used as positive control (considered as 100% cell death). The plate was shaken for 1 min, and the absorbance of formazan in each well was measured in a microplate reader at 570 nm. The percentage of cytotoxicity compared with the negative control  was calculated according to the following equation:The cellular viability assay described in ,22 was u50).The plot of percent cytotoxicity versus sample concentration was used to calculate the extract concentration that killed 50% of the cells  for 6 h and then exposed to 250 μM H2O2 for 1 h. The cytotoxic effect was calculated as a percentage of viability when compared with untreated cells (negative control considered as 100% of viability) and cells treated with 250 μM H2O2 for 1 h (positive control considered as 100% of cell death) [H2− generation inside the viable cells according to the method in [2O2 for 1 h (positive control: considered as massive absorbance). Following 3 h incubation, the supernatants were removed, and the formation of formazan product was solubilized in 100 µL DMSO. After mixing the contents in each well, the absorption of colored formazan solution was read at 620 nm using a Varioskan LUX multimode microplate reader .The reduction of NBT to insoluble blue formazan was used as a probe for Oethod in  with sli2. After the washing step with DPBS, the cells were treated with 200 µM of H2O2 for 2 h to undergo intracellular oxidation. Untreated cells were negative control (considered as moderate absorbance) and cells treated with 200 μM H2O2 for 1 h were positive control (considered as massive absorbance). Fluorescence intensity was measured using the Varioskan LUX multimode microplate reader  at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.To investigate the formation of ROS inside SHSY5Y cell line with and without plant extracts, 2,7-dichlorodihydrofluorescein diacetate (DCFHDA) was used . BrieflySHSY5Y cells were incubated separately with different volumes of DMSO (solvent used for solubilizing plant extracts) to check the viability of cells.A MLE sample with a concentration of 101.23 mg/mL in 50% methanol was prepared, ultrasonicated for 20 min, and centrifuged at 10,000 rpm at 4 °C for 10 min. The supernatant was filtrated with 0.2 µm nylon membrane and injected into liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer (LC–QTOF/MS) . The liquid chromatographic system consisted of a binary pump and an online vacuum degasser connected to a Dual AJS ESI source mass spectrometer . Full-scan mode was used from m/z 100 to 1700. Zorbax Eclipse Plus column   was used for the analysis. Formic acid 0.1% in distilled water (solvent A) and 100% acetonitrile (solvent B) were used as the mobile phase-gradient elution as follows: 98% A, 0–2 min; 90% A, 2–25 min; 85% A, 25–40 min; 80% A, 40–48 min; 75% A, 48–68 min; 70% A, 68–80 min; 50% A, 80–85 min; 0% A, 85–90 min; 98% A, 90–100 min. Peaks were detected at wavelengths of 254 and 280 nm. The MS spectra were acquired in both positive and negative ion modes, auto MS/MS. The mass fragmentations were identified using the spectrum database for organic compounds in the METLIN database .n = 3). Data were reported as mean ± SD values. Analysis of variant (ANOVA) was used to evaluate the differences among the treatments by Duncan’s multiple range tests (DMRTs) with p value of 0.05 using SPSS Statistics Base version 19 program for Windows . All investigations were conducted in this study in triplicate  and single electron transfer (SET) mechanisms [In the present study, the antioxidant capacities and neuroprotective activities of the four selected Asian plants—chanisms . VSE gavchanisms ,27,28, wchanisms . Table 1chanisms . Meanwhichanisms . Althougn = 3) while for DPPH and neuroprotection results expressed in cell viability percentage, (μmol/g): μmol of Fe (ΙΙ)/g of dry weight plant extract). ND (not determined): extract excluded from viable system (SHSY5Y) experiments due to high cytotoxicity. a, b, c, d, e, f, g are significant differences with p-value (<0.05). MLE: M. oleifera leaves extract; VSE: V. Vinifera seeds extract; PLE: P. ginseng leaves extract; PRE: P. ginseng rhizomes extract; ALE: A. galanga leaves extract; ARE (PYL): A. galanga rhizomes extract ; ARE (BL): A. galanga rhizomes extract (brown layer).The data are expressed as mean ± SD  cell line was performed. This neuronal cell line was selected for the following reasons: (a) this cell line is acquired from a human origin, a source from which primary neurons are difficult to obtain (b) cells have been proposed as a useful model for studying the effect of free radicals formation and scavenging in the human neurons as they possess many biochemical and functional properties of neurons (c) the cell population is reasonably homogenous, which hypothetically translates into reproducibility and (d) these cells are oncogenic and their proliferation to yield a sufficient quantity for investigations can be accomplished.50) of the extracts are summarized in 50 values of 2.7 ± 0.2, 2.53 ± 0.1, and 2.43 ± 0.1 mg/mL, respectively, whereas ARE (PYL) showed the most cytotoxic effect to SHSY5Y cells with an IC50 of 0.34 ± 0.02 mg/mL. The data of ARE (BL) showed high toxicity on the neuroblastoma cell line, even in low concentrations. After testing the cytotoxicity of each extract and optimizing the lethal concentration of H2O2 on SHSY5Y cell line, the extracts were investigated for their neuroprotection activity in the presence of H2O2. It was found that PRE provided the greatest amount of neuroprotection, with 51% of viable cells, followed by MLE and ARE (PYL), with 44% and 40% of viable cells, respectively. Several studies reported that ginsenosides in PRE can support brain function, prevent neuroinflammation and oxidative stress, and inhibit or weaken various neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease (HD), and traumatic brain injury [Before investigation of their neuroprotection activities, all of the studied plant extracts were tested for cytotoxicity on SHSY5Y cells by MTT assay. The results of 50% inhibition concentration , followed by VSE (0.98 ± 0.08 mg/mL). ALE and PRE showed less reduction activity—1.24 ± 0.05 and 1.22 ± 0.06 mg/mL, respectively. ARE (PYL) and PLE showed the lowest O2− radical scavenging activities—1.62 ± 0.1 and 1.65 ± 0.04 mg/mL, respectively. 2−) and treated cells with MLE (light colored with low O2−).Using the NBT test, we assayed different plant extracts for their capacity to protect the SHSY5Y cell line from O2− and intracellular ROS were determined using Pearson coefficient correlation. The strongest and best correlation was found between DPPH and IC50 of NBT reduction activity with a value of r = −0.83, p < 0.05; the results of the correlation established that extracts that possess higher quenching DPPH values are able to scavenge O2− with lower IC50 in the NBT test, as shown in p-value > 0.05).Correlations between the free radical scavenging effect of tested extracts  and ALE) outside the cells by DPPH and FRAP tests and inside the viable cells by scavenging O50 values of 0.71 ± 0.08, 0.83 ± 0.08, and 0.96 ± 0.1 mg/mL, respectively, exhibited the best ROS scavenging activity. VSE (1.24 ± 0.06 mg/mL) and PLE (1.34 ± 0.06 mg/mL) showed less effectiveness in decreasing intracellular ROS levels, whereas ALE (2.48 ± 0.03 mg/mL) showed the lowest antioxidant activity in SHSY5Y cells. Moreover, SHSY5Y cells treated with DMSO showed no effect on the cells. The unique activity of MLE might be due to the presence of potent compounds that are commonly known to have antioxidant efficiency, such as quercetin derivatives, isorhamnetin glucoside, kaempferol, luteolin and its derivatives, ascorbic acid derivative, diosmetin, apigenin and its derivatives (saponarin), hydroxytyrosol 1-O-glucoside (HT), and esculetin, as shown in To detect the capacity of the obtained plant extracts to scavenge intracellular levels of ROS in SHSY5Y cells, DCFHDA was applied as a fluorogenic substrate. This substrate was converted to highly fluorescent DCFH by ROS and was monitored by the spectrophotometer. From the data mentioned above, this extract ranked as first or second place in all experiments; the revealed data established that ethanolic MLE extract is pharmacologically more active than other tested extracts, which might be because of the synergistic effects of various potent components present in the whole extract.2O2 and preventing cell damage by oxidative stress. It also contained various effective phenolic compounds that support a powerful antioxidant activity, including apigenin 7-rhamnosyl-(1→2)-galacturonide, cartormin, kaempferol 4′-glucoside, hesperetin, and isorhamnetin 3-(6″-acetylglucoside).Taken together, MLE, grown in Thailand, is interesting as a potential neuroprotective agent with further applications due to its low cytotoxicity and high antioxidant activities. LC–QTOF/MS analysis  of MLE iMLE requires more attention, as it plays an important and unique biological role as an ecofriendly and non-cytotoxic antioxidant agent compared to other tested extracts in all tests.A. galanga, V. vinifera, M. oleifera, and P. ginseng cultivated in Thailand were selected in the present study.A variety of phytochemicals have been reported to prevent the risk of numerous diseases, including neurodegenerative diseases. In Asian countries, many researchers are seeking bioactive compounds from edible plants or herbs for the prevention or treatment of neurodegenerative diseases. MLE showed high antioxidant activities (DPPH and FRAP assays) with low cytotoxicity to SHSY5Y cell lines compared to the other studied plants. Its mechanism of action for neuroprotective effect on SHSY5Y cells is probably due to its high level of polyphenolic and other antioxidant compounds, and it possesses the ability to scavenge free radicals or activate the cellular antioxidant system. LC–QTOF/MS analysis confirmed that MLE consists of phenolic compounds, which are a good source of antioxidants. The determination of pure bioactive compounds involved in neurodegeneration contained in MLE should be the subject of further investigation.


A 35-year-old obese female patient presented to the emergency department (ED) endorsing symptoms of generalized weakness, dyspnea, and myalgia. Vitals on admission revealed hypotension, tachycardia, and a low-grade fever. Physical examination was unremarkable and was negative for any upper right quadrant tenderness or jaundice. Laboratory results revealed an elevated leukocytosis with a predominantly elevated neutrophil count, an elevated lactate dehydrogenase, aminotransferases, and an elevated anion gap. Sepsis protocol was initiated. Blood cultures revealed Group F Streptococcus. A chest x-ray for localization of the primary infection source was significant for an incidental hypodense liver mass. A follow-up magnetic resonance imaging (MRI) without contrast revealed a left hepatic multiloculated enhancing lesion prompting the diagnosis of beta-hemolytic Group F Streptococcus pyogenic hepatic abscess (PHA). This unusual case seeks to inform that an obese patient  with systemic signs  should warrant careful monitoring as well as the inclusion of pyogenic liver abscess in the differential workup as our patient’s PHA was found incidentally on a chest x-ray. Appropriate management via sonographic guided drainage was initiated and systemic antibiotics were administered in both inpatient and outpatient settings, resulting in complete resolution of the hepatic abscess over the course of a month. A pyogenic hepatic abscess (PHA) most commonly presents with symptoms of fever, right upper quadrant (RUQ) pain, and jaundice. Common risk factors include, but are not limited to, intravenous drug use, diabetes mellitus (DM), proton pump inhibitor use, and more . PHAs ar2) with no travel history and not on any medications presented to the ED with symptoms of generalized weakness, body aches, and difficulty in breathing. Vital signs recorded blood pressure of 84/55 mmHg, a heart rate of 128 beats per minute, temperature of 100.6℉ (38.1℃), respiration rate of 18 breaths per minute, and oxygen saturation of 98%. Initial laboratory showed an elevated leukocytosis of 19.1 x 103/µL  with a predominant neutrophil count (ANC) of 17.6 x 103/µL . The patient’s lactic acid was elevated to 4.1 mmol/L , as well as her alanine aminotransferase level (ALT) of 71 U/L , aspartate aminoransferase (AST) level of 56 U/L , and an anion gap of 24 mmol/L . A portable chest x-ray (CXR) was performed and revealed pulmonary vascular congestion. Three out of the four systemic inflammatory response syndrome (SIRS) criteria were met leading to the initiation of the sepsis fluid protocol. Intravenous (IV) norepinephrine 5 mcg/min was also started for additional blood pressure support. Repeat labs revealed an up-trending leukocytosis to 22.5 x 103/µL. The patient was being treated for sepsis pending work-up and started on IV vancomycin and IV piperacillin/tazobactam for broad-spectrum coverage as per infectious disease (ID). After stabilization, the patient was admitted to the intensive care unit (ICU) for further management. Results from the blood cultures showed Group F Streptococcus with sensitivity to ampicillin, ceftriaxone, penicillin, clindamycin, cefotaxime, and vancomycin. The antibiotic regimen was then narrowed to IV ceftriaxone 2g daily. A repeat CXR done on hospital day 7 revealed a hypodense liver mass. Further workup via magnetic resonance imaging (MRI) without contrast on day 8 revealed a left hepatic multiloculated enhancing lesion measuring 8.9 cm x 6.1 cm x 5.1 cm . However, status post drainage the patient developed rigors and a maximum fever of 101℉ (38.3℃) and a rapid response was called until the patient was stabilized. Liver abscess cultures were negative for Entamoeba histolytica, ova, and parasites but positive for Group F Streptococcus constellatus. Per os (PO) metronidazole 500 mg was then added to the patient’s antibiotic regimen. Repeat computerized tomography (CT) scan with contrast on day 17 revealed a mild decrease in liver abscess size from 7.1 cm x 6.6 cm to 5.8 cm x 4.0 cm. As per surgery recommendations, the drain was kept in place and a CT scan was scheduled in two weeks. On day 19, WBC levels were down trended, and the patient was discharged on home infusion antibiotics via a peripherally inserted central catheter (PICC) line on a regimen of IV cefepime 2 g every eight hours, IV vancomycin 1g every 24 hours, and PO metronidazole 500 mg every eight hours for a minimum of three weeks as per ID recommendation. However, on the same evening after discharge, the patient reported feeling chills and returned to the ED. She was found to have a maximum temperature of 102.7℉ (39.2℃) and a WBC level of 23.1 10As described in previous literature, the risk of PHA development is increased with underlying risk factors such as IV drug abuse, DM, malignancy, other comorbidities . FurtherWe present a unique case of an asymptomatic incidental PHA in a patient with obesity as their only co-morbidity. We aim to enlighten clinicians on such a case to ensure a comprehensive differential workup is done to rule out PHA even with a lack of typical hepatic abscess symptoms such as RUQ pain, fever, and jaundice.


Cassia fistula stem bark in STZ-diabetic animals. Novel triterpenoid compounds  were treated to STZ-administered diabetic animals at a concentration of 20mg/kg body weight orally for 60 days to assess their effects on plasma glucose, plasma insulin/C-peptide, serum lipid markers and the enzymes of carbohydrate metabolism, glucose oxidation and insulin signaling molecules. Oral administration of novel triterpenoid compounds to STZ-diabetic animals significantly decreased (p < 0.05) the plasma glucose concentration on the 7th, 15th, 30th, 45th and 60th daysin a duration-dependent manner (p < 0.05). Plasma insulin (p < 0.0001)/C-peptide (p < 0.0006), tissue glycogen (p < 0.0034), glycogen phosphorylase (p < 0.005), glucose 6-phosphatase (p < 0.0001) and lipid markers were significantly increased (p < 0.0001) in diabetic rats, whereas glucokinase (p < 0.0047), glycogen synthase (p < 0.003), glucose oxidation (p < 0.001), GLUT4 mRNA (p < 0.0463), GLUT4 protein (p < 0.0475) and the insulin-signaling molecules IR mRNA (p < 0.0195), IR protein (p < 0.0001), IRS-1 mRNA (p < 0.0478), p-IRS-1Tyr612 (p < 0.0185), Akt mRNA (p < 0.0394), p–AktSer473 (p < 0.0162), GLUT4 mRNA (p < 0.0463) and GLUT4 (p < 0.0475) were decreased in the gastrocnemius muscle. In silico analysis of C1–C3 with IRK and PPAR-γ protein coincided with in vivo findings. C1–C3 possessed promising antidiabetic activity by regulating insulin signaling mechanisms and carbohydrate metabolic enzymes.Here, we identified the mechanisms of action of antidiabetic activity of novel compounds isolated from  Skeletal muscle is the key site for insulin-activated glucose consumption in vivo. Skeletal muscle insulin resistance is a primary and important phenomenon for the progression of type-1 diabetes mellitus (T1DM) . About 8Cassia fistula (C. fistula) stem bark.Herbal compounds have been utilized since early times to treat T1DM . About 9Tetracyclic triterpenoids reported in medicinal plants are widely used in managing the ailments of diabetes . They haC. fistula stem bark possessed hypolipidemic and hypoglycemic activity in streptozotocin (STZ)-administered diabetic animals. It also enhanced glucose oxidation through GLUT4 in the skeletal muscle [Previous findings from our laboratory showed that novel catechin isolated from l muscle . As an e1HNMR and 13C NMR  to diabetic rats reduced the plasma glucose concentration significantly on the 7th, 15th, 30th, 45th and 60th days in a duration-dependent manner. Furthermore, 60 days of treatment effectively reduced the plasma glucose concentration to a level equal to that of the normal control rats and insulin-treated diabetic rats . Hence, Plasma insulin and C-peptide concentrations were decreased significantly in STZ-administered diabetic animals. Diabetic rats treated with novel triterpenoids (C1–C3) and insulin did not have increased plasma insulin and C-peptide concentrations compared to the control a,b.Tissue glycogen content in the liver and skeletal muscle of control and diabetic animals are shown in The activity of the enzyme glycogen synthase, which is involved in glycogenesis, was decreased significantly in STZ-administered diabetic animals c. The acDiabetic animals exhibited a significant increase in the levels of serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL) and a significant reduction in the level of high-density lipoprotein cholesterol (HDL) a–d. OralIn diabetic animals, oxidation of glucose was significantly decreased compared to control . However612) were decreased significantly in STZ-administered diabetic animals, but novel compound treatment significantly raised the same (Insulin receptor (IR), IRS-1 mRNA and their phosphorylation  were reduced significantly in diabetic animals  and PHE 1151 (NH…O=C), whereas compound 3 had three H bond interactions, with ALA 1080 (OH…O=C), PHE 1151 (NH…O=C) and ASP 1083 (OH…O=C). PPAR-γ also showed hydrogen bonding interactions with all three compounds. The OH group of compound 1 interacted with the NH group of the amino acid GLN 283. The OH group of compound 2 had two H bond interactions, with ASP 260 (OH…O=C) and SER 342 (OH…O=C), and compound 3 had two H bond interaction, with 260 (OH…O=C) and GLU 343 (OH… O=C).Molecular docking helped to discover an appropriate ligand for the receptor by generating an optimal protein–ligand bound conformation. All docking results were monitored by scoring functions that predicted the binding affinity of a ligand in a particular docked pose . High glDuring summer, fresh bark of C. fistula was collected from Kodaikanal, Tamil Nadu, India. The species was identified and authenticated at the Department of Botany, Holy Cross College, Tiruchirapalli, and the voucher specimen was deposited in the herbarium of the Department.The shade-dried plants were powdered, and 1 kg of powder was extracted sequentially using hexane, ethyl acetate and methanol in a Soxhlet apparatus. The extracts were evaporated to dryness under reduced pressure in a rotary evaporator. The yield of hexane extract was 17.8 g, that of ethyl acetate extract was 16.6 g and that of methanol extract was 20.1 g. The dry residues of these crude extracts were stored at 4 °C for further use. The ethyl acetate extract was further chromatographed on a silica gel column  and then successively eluted with a continuous gradient from 100% hexane, 95% hexane and 5% ethyl acetate to10% ethyl acetate. The fractions were collected, and each fraction was spotted on a precoated Silica gel 60 F254, 0.25 mm thick thin layer chromatography (TLC) plate (Merck). Fractions with similar Rf values in the TLC pattern were pooled together into 18 fractions. Fractions 2, 8 and 9 gave single spots in TLC. The compounds were subjected to nuclear magnetic resonance (NMR) and mass (MS) and infrared (IR) spectral analyses  for struHealthy adult male albino Wistar rats (weighing 150–220 g) were used in the present study. Animals were maintained under specific temperature (21 ± 2 °C) and humidity (65 ± 5%) with a constant 12 h light and 12 h dark schedule and fed with a standard pelleted diet  and clean drinking water ad libitum.In the experimental animals, after overnight fasting, diabetes was induced by single intraperitoneal injection of60 mg/kg body weight STZ  in a freIn this study, a total of 54 animals were divided into 9 groups of 6 animals each. Group 1: vehicle control , 1 mL/kg body weight); Groups 2–4: normal control rats treated with novel triterpenoids (20 mg/kg body weight); Group 5: STZ-induced diabetic rats; Groups 6–8: diabetic rats treated with novel triterpenoids (20 mg/kg body weight); Group 9: insulin-treated diabetic animals (3 IU/kg body weight). The dosage of compounds was fixed from the acute toxicity tests reported in our previous study, which were filed for patent ,44,45. TSerum blood glucose concentration was determined by a commercially available glucose kit . Results were expressed as mg/dL.125I-labelled human insulin antibody, which has cross-reactivity with rat insulin. Plasma C-peptide level was assayed by a radioimmunoassay kit procured from Missouri, USA. Results were expressed as µmol/mL.Radioimmunoassay was used to test plasma insulin using a kit method . The kit included human insulin as standard and g. The supernatant was decanted, and the tubes were allowed to drain on a filter paper for few minutes. The precipitate was dissolved in 1 mL of distilled water, precipitated with 1 mL of 95% ethanol, centrifuged and drained as stated before. The precipitate was dissolved in 5 mL of distilled water, and 10 mL of 0.2% anthrone reagent was added under ice-cold conditions. Five milliliters of distilled water and a series of standards with final volumes of 5 mL were treated with the anthrone reagent and subjected to the same procedure. The tubes were covered with glass marbles and heated for 10 min in a boiling water bath. The contents were cooled immediately, and the color developed was read at 680 nm. The amount of glycogen was expressed as g/100 g of wet tissue.Glycogen was estimated in the liver and skeletal muscle of the control and treated animals . Five miAccording to the standard methods , glucokiSerum TC, TG, HDL and LDL were measured as per the manufacturer’s instructionsusing akit procured from Diagnostic kit . Results were expressed as mg/dL.14C-glucose. Briefly, 10 mg tissue was kept in a 2 mL ampoule containing 170 µL Dulbecco’s modified Eagle’s medium (DMEM), pH 7.4, 10IU penicillin and 0.5 μCi 14C-glucose. The ampoules were then aerated with a CO2trap containing a gas mixture. The closed system with the CO2 trap was then kept in an incubator at 37 °C. The CO2trap was replaced after 2 h. After removing the second trap and releasing any remaining CO2 from the sample, 0.1 mL of NH2SO4 was applied to prevent further metabolism. The process was closed 1 h before the third once again, and the final trap was removed. Finally, all the CO2 traps were placed in a scintillation vial containing10 mL scintillation fluid and counted in a β-counter. Results of 14CO2 released/10 mg tissue were expressed as CPM.Oxidation of the glucose in the skeletal muscle of the control and treated animals was measured  using 14g for 15 min at 4 °C. The aqueous phase (upper layer) was carefully transferred to a fresh microcentrifuge tube, and isopropanol was added in equal quantity, vortexed for 15 s and placed on ice for 10 min. Then, the contents were centrifuged for 10 min at 4 °C at 12,000× g, the supernatant was discarded, and the RNA pellets were washed by quick vortexing with 1 mL of 75% ethanol. The total RNA was quantified using a spectrophotometer procedure [Total RNA was isolated using a TRIR kit . Briefly, 1 mL TRIR was added to 100 mg of tissue and homogenized. The contents were transferred into a microcentrifuge tube, and 0.2 mL chloroform was added, vortexed for 1 min and kept at 4 °C for 5 min, then centrifuged at 12,000× rocedure . The totrocedure . BrieflyTissue samples from the gastrocnemius muscle were prepared as per the standard protocol . Then, tTyr612, Akt 1/2/3 and p-AktSer473, GLUT4  and β-actin monoclonal (at 1:1000 dilution) using 5% nonfat dried milk.Lysate proteins (50 μg/lane) were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a membrane by electroblotting  of polyvinylidene difluoride (PVDF). The membrane was blocked and tested with primary antibodies of IR-β, IRS-1, p-IRS-1Upon incubation the membrane was washed with tris-buffered saline containing Tween20 (TBS-T) three times (5 min each). Then, washed with TBS-T, horseradish peroxidase (HRP)-conjugated rabbit-anti-mouse or goat-anti-rabbit antibodies  were incubated with the membrane for 1 h. Using an enhanced chemiluminescence detection system , the specific signals were detected, and using Chemidoc, the protein bands were captured and quantified . Afterward, the membrane was reprobed with β-actin antibody (at 1:5000). In the present study, rat β-actin was used as a loading control.Statistical analysis was conducted using the SPSS software package version 17.0. The values were evaluated using a one-way variance analysis (ANOVA) followed by the multiple-range test (DMRT) .http://www.rcsb.org, accessed on 25 October 2021) with the accession IDs1IRK and 1ZGY, respectively. Two types of facilities  were accessible in the “protein preparation wizard”of the Schrodinger software . First, the chemical accurateness was ensured; then, hydrogen atoms were added, and side chains that were neither near the active site nor participatoryin the creation of salt bridges were neutralized by using the optimized potentials for liquid simulations–all atom (OPLS–AA) force field .The crystal structures of IRK and PPAR-γ were downloaded from PDB  Schrodinger, LLC. New York). These structures were changed to the.mae format, suitable bond orders were given for every structure and tautomers for all of these ligands were produced and optimized. Partial atomic charges were computed using the OPLS2005 force field.The three selected novel compounds were docked into the active sites of IRK and PPAR-γ using the Glide XP module . The shapes and properties of the target proteins were shown on a grid by many sets of fields that gradually gave more perfect scoring to the ligand poses. The grid wassetto 12 Å × 12 × Å 12 × Å for the analysis of docking study. Geometric or hydrogen-bonding restraints were not introduced for substrate docking. Docking studies were carried out with the default parameters.C. fistula controlled hyperglycemia through activation of IRS-1/Akt-mediated insulin signaling mechanisms and through regulation of carbohydrate metabolic enzymes in the skeletal muscle and liver. Hence, we concluded that supplementation of triterpenoids could be a promising therapeutic approach in the management of diabetes. Clinical trials engaging such triterpenoids could be of intensive interest and beneficial for the management of diabetes.Our present findings clearly showed that the novel triterpenoid compounds from


Thus, the OLFM4/HIF-1α axis might be a target signaling pathway for developing novel drugs to treat HCC.Hepatocellular carcinoma (HCC) is one of the primary types of cancer that claims many lives worldwide, and its incidence continues to increase. Conventional therapies against liver cancer are inadequate, and the pathogenesis of HCC remains unclear. Thus, not only are more effective therapies to treat HCC required but also identification of the key genes involved in its pathogenesis is important for developing such therapies. This study found that olfactomedin 4 (OLFM4) level is higher in HCC patients than in healthy individuals. Furthermore, HCC patients also have higher messenger ribonucleic acid (mRNA) expression level in HCC tissues than in liver paracancerous tissues. OLFM4 has high predictive capacity as a biomarker for HCC and closely correlates to tumor size. It is confirmed that OLFM4 contributes to cancer cell proliferation, and HIF1 Liver cancer is the fourth leading cause of cancer-related deaths after lung, colorectal, and stomach cancer, and its global incidence continues to increase . HepatocOlfactomedin 4 (OLFM4) belongs to the olfactomedin family, which is also known as the human granulocyte colony-stimulating factor-stimulated clone 1(hGC-1) . Initialα axis was found to be involved in the regulation of hypoxia-induced invasion, epithelial-mesenchymal transition, and chemotherapy resistance in non-small-cell lung cancer [α is a subunit of a heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), which plays an essential role in cellular response to systemic oxygen levels in mammals [α is closely involved in the pathogenesis of cancer. HIF-1α and GATA3 form a complex that enhances cancer cell invasiveness [α is a potential therapy for alleviating chemoresistance to enhance the efficacy of chemotherapy in colon cancer [α axis could improve hypoxia-induced invasion, epithelial-mesenchymal transition, and chemotherapy resistance of non-small-cell lung cancer [α. Thus, this study proposes OLFM4 as a potential biomarker and therapeutic target for HCC and provides critical information for studying pathogenesis and developing novel drugs against HCC.The OLFM4/HIF-1g cancer . HIF-1α  mammals . HIF-1α siveness . Targetin cancer . Interesg cancer . Therefo3 copies per mL, as well as abnormal concentration of serum alanine amino transferase. Confirmation of chronic HCV infection was used for qualitative HCV-RNA measurement, and more than 1 × 103 copies of HCV-RNA in the serum were confirmed to be positive. Healthy controls were identified as without liver or other systematic diseases or HBV markers , as well as normal concentrations of liver function enzymes. The study was approved by the institutional ethics review committee at the Bayannur Hospital. Informed consent was obtained from all participants based on each committee's regulations.Beginning from May 2016 to May 2020, we recruited 100 HCC patients and 100 healthy controls from the Bayannur Hospital. HCC patients were diagnosed using histopathological analysis. Among 100 HCC patients, 80 received surgery and 20 underwent interventional therapy. HCC was diagnosed according to immunohistochemistry based on the AASLD guidelines. Patients who received radiotherapy or had a history of other solid tumors were excluded. NASH was confirmed based on the histopathology of liver biopsy samples and supported by imaging evidences, such as CT and liver ultrasound. Chronic HBV infection was confirmed by HBsAg presence in the last 6 months with an HBV DNA concentration to >1 × 10Serum samples were obtained from patients who were diagnosed with primary HCC at the Bayannur Hospital. Serum from healthy individuals was simultaneously collected at the hospital as control samples. Serum samples were collected under institutional approval. The serum was centrifuged, aliquoted, and stored at −80°C for diagnosis utilization.The concentrations of OLFM4 were detected using ELISA. ELISA kits were purchased from Abcam . ELISA experiments were performed in strict accordance with the manufacturer's instructions.Total RNA was extracted from HepG2 cells and liver biopsy using Trizol reagent from Invitrogen . cDNA synthesis was performed using the Maxima Universal First Strand cDNA Synthesis Kit from Thermo Scientific . RT-qPCR reactions were performed with FastStart Universal SYBR Green Master (Rox) from Roche . The experiments were performed according to the manufacturer's instructions. For IHC analysis, cancer and paracancerous tissues from liver patients were formalin-fixed, followed by embedding by using the paraffin method. Subsequently, paraffin blocks were prepared into slides, followed by the IHC process using standard instructions. Anti-OLFM4 antibody  was used to probe the slides, which were then visualised using DAB + as a chromogen.Human liver carcinoma cells (HepG2) were purchased from ATCC (ATCC HB-8065). HepG2 cells were maintained in Dulbecco's modified Eagle medium  containing 10% fetal bovine serum , 100 units/ml penicillin, and 0.1% (w/v) streptomycin  at 37°C in a humidified atmosphere of 5% CO2.α against OLFM4 were synthesized by Thermo Fisher Scientific. SiRNAs against OLFM4 and HIF-1β-actin  at 4°C overnight, followed by two washes with TBST. Thereafter, membranes were incubated with the appropriate secondary antibodies at room temperature for 1 h and washed thrice with TBST. Protein bands were visualised using chemiluminescence .We used the conventional protocol for western blotting (WB). Briefly, cells were lysed for total protein isolation using RIPA lysis buffer . The protein concentration was determined using the Bradford assay. Equal amounts of total protein were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes, and blocked with 5% skim milk powder at room temperature for 1 h. Subsequently, the PVDF membranes were washed with TBST containing NaCl, Tris-HCl, and Tween-20 and incubated with primary antibodies against target proteins, including OLFM4  and t-test and one-way ANOVA. The levels of mRNA expression between cancer tissues and normal tissues were analyzed by the t-test. All analyses related to patient survival were tested by Kaplan–Meier survival analysis (log-rank method). A P < 0.05 was regarded as statistically significant.To examine the differences between groups, we used Student's P=0.0778). Among female patients, BMI was 20.67 ± 0.27, and it was 20.49 ± 1.86 among male patients (P=0.4683). There were 20 HBV-infected female patients and 31 HBV-infected male patients (P=0.895); 6 HCV-infected female patients and 7 HCV-infected male patients (P=0.668); and 5 NASH female patients and 18 NASH male patients (P=0.059). For female patients, the OLFM4 level in blood was 44.06 ± 1.67 (U/L), and it was 43.16 ± 2.23 (U/L) (P=0.7815) for male patients. For female patients, the tumor size was 2.683 ± 0.30 , and it was 43.16 ± 2.23  (P=1.72 ± 0.14) for male patients.P < 0.001). To further demonstrate, the mRNA expression level of OLFM4 in liver paracancerous and cancer tissues was measured. The mRNA expression level of OLFM4 in HCC tissues was remarkably higher than that in liver paracancerous tissues (P < 0.001). OLFM4 staining was observed in HCC tissues but not in liver paracancerous tissues (P < 0.001). Thus, OLFM4 expression level was significantly upregulated in HCC patients.To test the potential of OLFM4 as a biomarker of HCC, OLFM4 blood level was measured using ELISA. OLFM4 level in HCC patients was significantly higher than in healthy individuals (P < 0.0001) and 0.8844 (P < 0.0001), respectively.Receiver operating characteristic (ROC) analysis was performed to determine the diagnostic value of OLFM4 expression for HCC . It indiR2 = 0.4646, P < 0.0001)   . OLFM4 m 0.0001) . CollectTo investigate the effects of OLFM4 on HCC, a human liver carcinoma cell line  was used. First, two siRNAs against OLFM4 were constructed, and both siRNAs showed knockdown effects on OLFM4 in HepG2 cells, while the second siRNA showed better knockdown efficiency . Knockdoα was found to be the downstream gene of OLFM4, which encouraged us to study whether HIF-1α is involved in the regulation of OLFM4 in HCC. Two siRNAs against HIF-1α were synthesized and transfected in HepG2 cells, which indicated a significant knockdown (α significantly alleviated the promotion of OLFM4 on HepG2 proliferation (α involves the regulation of OLFM4 on HCC cell proliferation.HIF-1nockdown . It was feration . Thus, wα is involved in the regulation of OLFM4 on HCC cell proliferation.HCC has been become one of most severe types of cancer claiming many lives worldwide . AlthougCancer incidence varies greatly depending on gender. For example, it was found that gender differences existed in cancer-associated venous thromboembolism . Yang etHCC has been found to induce the expression modification of a large body of genes. Zhang et al. used bioinformatics analysis to identify several key genes and pathways in HCC, including GMPS, ACACA, ALB, TGFB1, KRAS, ERBB2, BCL2, EGFR, STAT3, and CD8A . Similarα induced apoptosis [α is involved in the regulation of OLFM4 on hypoxia-induced invasion, epithelial-mesenchymal transition, and chemotherapy resistance in non-small-cell lung cancer [α is involved in the regulation of OLFM4 in terms of HCC cell proliferation (α axis might be a target signaling pathway for developing novel drugs to treat HCC.Seeking a therapeutic target is vital for developing effective drugs to treat cancer. This study found that OLFM4 promoted HCC cell proliferation . In gastpoptosis . Consistpoptosis . Interesg cancer . This stferation . Thus, tα is involved in this process. We believe that the OLFM4/HIF-1α axis might be a target signaling pathway for developing novel drugs to treat HCC.In summary, OLFM4 level is higher in HCC patients than in healthy individuals, and mRNA expression level is higher in HCC tissues than in liver paracancerous tissues. OLFM4 has high predictive capacity as a biomarker for HCC and is closely correlated to tumor size. Most importantly, we confirmed that OLFM4 contributes to cancer cell proliferation, and HIF-1The main findings and implications of the work are clearly explained, highlighting its importance and relevance.


MoYPD1 in the rice blast fungus Magnaporthe oryzae is subjected to an increased structural variation and amplified putative isoforms on a cDNA level. We then generated mutant strains overexpressing these isoforms, purified the products, and present here one previously unknown MoYpd1p isoform on a proteome level. Alternative splicing was found to be a valid molecular mechanism to increase the signal diversity in eukaryotic multistep phosphorelay systems.Different external stimuli are perceived by multiple sensor histidine kinases and transmitted by phosphorylation via the phosphotransfer protein Ypd1p in the multistep phosphorelay system of the high osmolarity glycerol signaling pathway of filamentous fungi. How the signal propagation takes place is still not known in detail since multiple sensor histidine kinase genes in most filamentous fungi are coded in the genome, whereas only one gene for Ypd1p exists. That raises the hypothesis that various Ypd1p isoforms are produced from a single gene sequence, perhaps by alternative splicing, facilitating a higher variability in signal transduction. We found that the mRNA of  Saccharomyces cerevisiae is perceived by the sensor histidine kinase (HK) Sln1p and transmitted through the histidine-containing phosphotransfer (HPt) protein Ypd1p to the response regulator Ssk1p [Alternaria brassicicola, Aspergillus nidulans, Botrytis cinerea, Fusarium oxysporum, and Neurospora crassa, the rice blast fungus Magnaporthe oryzae (anamorph Pyricularia oryzae) is an important model organism for researching the HOG pathway [Signal perception, transduction, and, consequently, the response to environmental changes is a prerequisite for fungal biology and controls essential pathways, such as stress response, differentiation processes, metabolism, and pathogenicity . The higor Ssk1p . Thus, Yor Ssk1p . In addi pathway ,9,10,11.M. oryzae contains ten genes for different sensor HKs , but only a single gene for a phosphotransfer protein is annotated, called MoYPD1  is experimentally proven [The genome of blFungi, ). It appy proven , whereasy proven ,12 , which has not been studied to a great extent for molecular mechanisms of signaling in fungal species. Alternative splicing is a molecular mechanism during the transcription process which has a vital role in eukaryotic gene regulation and increases the complexity of protein diversity . Differeisoforms .S. cerevisiae (1.8%), an unexpected higher number of splicing events were assumed in filamentous fungi, such as Shiraia bambusicola (38.88%) and Trichoderma longibrachitum (76.2%) [M. oryzae, two transcripts for MoYPD1  are predicted (EnsemblFungi), based on in silico data. Interestingly, a more detailed analysis of our RNAseq datasets revealed significantly more structurally different MoYPD1 transcripts [MoYPD1-associated sequences comprehensively on a DNA, RNA, and protein level. Furthermore, we generated a mutant strain overexpressing putative MoYpd1p isoforms and isolated and purified the products followed by LC-MS/MS analysis. In the end, we are able to present here experimental evidence of at least one new MoYpd1p isoform on a protein level.The broad spectrum of spliced mRNA and, consequently, the vast diversity of corresponding proteins illustrates the strong impact of AS on basic regulatory processes and signaling mechanisms within eukaryotic organisms. Whereas almost 95% of human multi-exon genes are alternatively spliced ,17, the  (76.2%) ,19,20. Inscripts . ConsequM. oryzae 70–15, respectively, ∆Moypd1 [3 (120 g), KCl (10.4 g), KH2PO4 (30.4 g), MgSO4 (10.4 g) × 7 H2O) and a trace element solution  × 7 H2O, H3BO3 (11 g), MnCl2 (5 g) × 4 H2O, FeSO4 (5 g) × 7 H2O, CoCl2 (1.7 g) × 6 H2O, CuSO4 (1.6 g) × 5 H2O, Na2MoO4 (1.5 g) × 2 H2O and Na2EDTA (50 g), at pH 6.5 adjusted by 1 M KOH). Minimal medium (pH 6.5) contains per liter: glucose (10 g), a 0.01% biotin solution (0.25 mL), nitrate salt solution (50 mL), a trace element solution (1 mL) and a 1% thiamine dichloride solution (1 mL).The fungal strain used in this study was  ∆Moypd1 . The strAll chemicals used were pro analysis quality unless otherwise stated.® plant mini kit , complying with the manufacturer’s instructions. The LunaScript® RT SuperMix Kit  was used for cDNA synthesis, following the manufacturer’s instructions. The RNA integrity numbers and DNA contaminations were assessed by an Agilent 2100 bioanalyzer  using the RNA 6000 Nano Kit (Agilent Technologies). High-quality RNA with RNA integrity numbers values above 8 were used for the transcriptome analysis.Mycelium of three-day-old liquid cultures was harvested, lyophilized, and ground into a fine powder under liquid nitrogen. Total RNA was isolated using the RNeasyA polymerase chain reaction (PCR) was performed in a total volume of 50 µL containing 5 µL of each primer (100 pmol/µL), 1 µL dNTPs (10 mM)) 2 µL cDNA, 10 µL 5× Phusion HF-Buffer, 0.5 µL Phusion Hot Start II DNA Polymerase  and 26.5 µL RNase free water to fill the reaction. The cDNA amplification was performed in a C1000 Touch Thermal Cycler , applying the following reaction conditions: initial denaturation for 30 s at 98 °C, 35 cycles consisting of denaturation at 95 °C for 10 s; annealing at 61 °C for 15 s, extension at 72 °C for 20 s, and a final extension step at 72 °C for 5 min. The PCR product was separated by electrophoresis in a 1% agarose gel , stained with ethidium bromide, and visualized using a QUANTUM-ST5-1100/26MX system .M. oryzae was isolated from mycelium of three-day-old liquid cultures (grown in CM at 26 °C and 120 rpm) using the DNeasy® Plant Mini Kit , following the manufacturer’s instructions for the purification of DNA from plants and filamentous fungi. The DNA manipulation followed standard procedures [® 10-β competent Escherichia coli strains (high efficiency) were used for the routine bacterial transformations and construction of plasmids.The DNA of ocedures . NEB® 10M. oryzae was conducted using Agrobacterium tumefaciens-mediated transformation. The detailed procedures followed those described previously [Fungal transformation of eviously .Agrobacterium-compatible vector pSJ + GFP(BAR) to generate the overexpression mutant ΔMoypd1/EF1::YPD1(TS) [−1 of the antibiotic in a minimal medium.The plasmid used for fungal transformation is based on a backbone of a binary YPD1(TS) . The mutΔMoypd1/EF1::YPD1(TS) was generated by the Gibson Assembly® cloning method [The plasmid used for fungal transformation of g method .pSJ + GFP(BAR/nTwinStrep) was generated by amplifying the fragment EF1prom from pSJ + GFP(BAR) with the primers SJ-632/SJ-633  as a spacer) was included in the primer SJ-634. The ScaI/BsrGI-restricted vector pSJ + GFP(BAR) was used as a backbone vector.2/SJ-633  and the pSJ + EF1::YPD1(BAR) was generated by using the BglII/BsrGI-restricted pSJ + GFP(BAR/nTwinStrep) as a backbone. The genomic sequence of MoYPD1 (Locus MGG_07173) was amplified from genomic DNA with the primers SJ-651/SJ-652.The 8674 bp expression-plasmid pSJ + EF1::YPD1(BAR) was finally used to transform ∆Moypd1 via Agrobacterium tumefaciens-mediated transformation, resulting in the mutant strains ΔMoypd1/EF1::YPD1(TS).The plasmid ΔMoypd1/EF1::YPD1(TS) was cultivated in 200 mL liquid CM in 500 mL glass flasks with one baffle for four days at 26 °C and 120 rpm. Mycelium was harvested by vacuum-filtrating the culture through filter paper , immediately frozen under liquid nitrogen, and freeze-dried. About 3 g of lyophilized mycelium of the expression strain ΔMoypd1/EF1::YPD1(TS) was used for the purification process. The samples were powdered with the TissueLyserII (Qiagen) using the Grinding Jar Set , according to the manufacturer’s instructions. The adapter sets were precooled at −80 °C for at least 2 h. The powdered mycelium was dissolved in binding buffer , 0.1%Tween 20), homogenized in an ultrasonic unit for 30 s, and then centrifuged for 45 min at 4 °C and 18,000 rpm. The supernatant was filtered , degassed, and applied to 1 mL Streptactin XT gravity-flow columns for affinity purification. Loading, washing steps, and elution were carried out according to the manufacturer’s instructions. The BXT elution buffer  was used for elution. Six fractions of 1 mL each were generated and selected fractions combined for the protein analysis.The strain Sixteen bands from the SDS-PAGE were excised and cut into 1 mm × 1 mm pieces and washed twice with 200 µL of 50 mM ammonium bicarbonate (AMBIC) in 50% acetonitrile (ACN) followed by a single wash with 200 µL pure ACN each by sonification for 5 min at room temperature, discarding the supernatant. The reduction of disulfide bonds was performed by the addition of 100 µL of 10 mM dithiothreitol in 50 mM AMBIC/50% ACN and incubation for 60 min at 56 °C. After discarding the supernatant, 100 µL of 55 mM iodoacetamide was added for alkylation of the reduced disulfide bonds by incubation for 45 min at room temperature, keeping the samples in the dark. The supernatant was discarded, and the gel pieces were washed twice with 200 µL of 50 mM AMBIC/50% ACN followed by a single wash with 200 µL pure ACN each by sonification for 5 min at room temperature, discarding the supernatant. The remaining ACN from the gel pieces was evaporated by warming the samples at 56 °C for 30 s. A proteolytic digest was performed by the addition of 2.5 µg Trypsin in 25 µL 50 mM AMBIC and incubation overnight at 37 °C. The resulting peptides were recovered by washing the incubated gel pieces twice with 50 µL 0.1% formic acid (FA) in 50% ACN and sonicating each for 15 min and combining the supernatants in a new sample tube. The peptides were frozen at −80 °C and lyophilized. The lyophilized peptides were reconstituted in 20 µL 0.1% FA for mass spectrometric analysis.A volume of 2.6 µL of the peptide mixture was injected in an ultra-high performance liquid chromatography nanoAcquity system (Waters Corporation) equipped with a HSS-T3 C18 1.8 μm, 75 μm × 250 mm reverse-phase column (Waters Corporation) for each measurement. The separation of peptides was performed at 55 °C using a 90 min gradient from 5 to 40% mobile phase B consisting of 0.1% FA/3% DMSO in ACN, while mobile phase A consisted of 0.1% FA/3% DMSO in water. The flow rate was set to 300 nL/min, and all separations were followed by washing the column with 90% mobile phase B and sufficient re-equilibration. After elution from the chromatographic column, the peptides were ionized by electrospray ionization and subjected to mass spectrometric analysis using a Synapt G2-S HDMS QTOF mass spectrometer (Waters Corporation) which was operated in data-dependent acquisition mode with a typical resolving power of R = 20,000. The top ten most intense peptide precursor m/z from MS1 spectra were selected for further fragmentation in the quadrupole, followed by measurement of the resulting fragment m/z as MS2 spectra. (Glu1)-Fibrinopeptide B was injected as a lock mass into the flow path every 30 s via the reference sprayer of the NanoLockSpray source for post-acquisition mass correction. All samples were measured in triplicates.M. oryzae reference proteome (UP000009058) downloaded on 17 March, 2021, with 12,791 entries; second, 173 common contaminants and, third, two amino acid sequences from putative YPD1 isoforms translated from possible open reading frames of cDNA sequenced previously. For confident peptide identification, all resulting peptides were filtered for 1% false discovery rate using a target decoy database, which corresponds to a −10 LgP score of 44.5 for each peptide spectrum match.The raw files were processed using PEAKS Xpro . The m/z error tolerance was set to 15 ppm for parent ion and 0.03 Da for fragment ion matches. Trypsin was specified as a digesting enzyme in specific mode, allowing up to one missed cleavage. Carbamidomethylation (+57.02 Da) on cysteine was set as a fixed modification, and one oxidation (+15.99 Da) on methionine as a variable modification was allowed. A combined FASTA file was used for the database search consisting of, first, the uniport https://www.ebi.ac.uk/pride/archive, accessed on 5 May 2021) partner repository with the dataset identifier PXD024832.The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE  and MGG_07173T0 (462 bp)) revealed the presence of 27 additional nucleotides in the 5′ direction. These findings indicated a novel transcript-based annotated isoform of MoYPD1 on the cDNA level. We predicted the open reading frames and translated them into amino acid sequences. MoYPD1 transcript in comparison to transcript MGG_07173T1 (Transcript 1) and MGG_07173T0 (Transcript 2), respectively.We strove to identify new isoforms of pathways . Towards data of  existingΔMoypd1/EF1::YPD1(TS), with an N-terminally attached Twin-Strep-tag® by using the loss of function mutant ΔMoypd1 as the parent strain to validate the new transcript-based in silico annotation on a protein level. The affinity tag was fused directly in front of one in silico predicted startcodon upstream in the 5′ untranslated region of the MGG_07173T1 and MGG_07173T0 annotations. That means, on the basis of this startcodon, isoforms would be transcribed and translated. After MoYpd1p-expression, affinity chromatography, and in-gel digestion, the peptide samples were analyzed by LC-MS/MS. As a result, two peptide fragments that were unique to the novel MoYPD1 transcript were confidently identified with a −10 LgP identification score above the quality threshold of 44.5. The first peptide MSEEEEENKK , but it did not completely restore the WT phenotype in vegetative growth [ΔMoypd1/EF1::YPD1(TS) phenotype resulted in the same observations as for ΔMoypd1/YPD1 in different stress-inducing ingredients, such as NaCl , sorbitol, and fungicide treatment. Up to now, we have isolated the new isoform only from mycelium of ΔMoypd1/EF1::YPD1(TS). Currently, we are on the way to look for different isoforms produced “signal-specific” or at different stages of the life cycle of the M. oryzae 70-15 strain.We next generated overexpression mutants of MoYpd1p, called sitivity . We showe growth . The ΔMoMoYPD1 transcript on a protein level  have been annotated. Despite the fact that MoYpd1p is known to be a phosphotransfer protein, knowledge of the putative alternatively spliced isoforms and their interactions with other pathway components is scarce. We present here the first report of a previously unknown protein isoform of MoYPD1, by capturing evidence on both a cDNA and protein level. The cDNA of the newly identified transcript contains 27 nucleotides more than the annotated transcripts. We were able to confirm two unique peptides (MSEEEEENKK and CESDSEENADK, MoYPD1 transcript by nanoUPLC-MS/MS. Of course, these additional N-terminal peptides could affect the 3D structure, folding, and, thereby, the phosphotransferase activity of MoYpd1p. Our results demonstrate clearly that more MoYpd1p isoforms exist than previously suggested, which may increase the signal diversity and provide the basis for further studies on signal transduction in fungal MSP systems. The discovery of the new isoform is consistent with recent studies which showed that AS events play a crucial role in the fungal kingdom [MoGRP1, for example, encoding a glycine-rich RNA-binding protein, was identified as a splicing factor, which is essential for the removal of introns in M. oryzae. The deletion of MoGRP1 resulted in a reduced stress response, virulence, mycelial growth, and conidiation [Verticillium dahlia has shown that approximately 50% of multi-exonic genes are subjected to AS regulation, with 90% of splicing events due to intron retention. In addition, the study showed that a gene can be transcribed via four different 5′-splice sites and two different 3′-donor sites into five mature mRNAs [Sclerotinia sclerotiorum. Isoforms merely expressed in planta by host-specific AS were identified in this phytopathogen. Thereby, colonization of diverse host families led to the accumulation of different transcripts and, consequently, altered protein domain structures, suggesting alternative functions of the isoforms during plant infection [Investigating signaling pathways to unravel the dogma of sensing multiple signals despite a limited number of pathway components might improve our current understanding of physiological processes in fungi. The phosphotransfer protein MoYpd1p is the key mediator within the phosphorelay system of the HOG pathway and enables signal transduction via phosphorylation and dephosphorylation in response to environmental changes. Despite MoYpd1p seeming to interact with multiple sensor HKs and at least one response regulator, only one coding gene . oryzae . That raed is AS . To date kingdom ,27. The idiation . Consequre mRNAs . Anothernfection .OsBWMK1, a MAPK family member, was detected in rice tissue. After various stress conditions, AS was shown to alter the domain architecture, affecting its subcellular localization but not its activity. Ypd1p homologs from Candida albicans and S. cerevisiae have previously been detectable in both cytoplasm and nucleus [M. oryzae, altering the subcellular localization of the protein rather than the protein function. Up until now, little research has previously dealt with the AS of phosphotransfer-protein encoding genes and the physiological purpose of different phosphotransfer protein isoforms. In the current study, we presented the first step to study AS as a molecular mechanism of signal transduction diversity and demonstrated insights to answer some of the fundamental questions in biology: how diversity is achieved in signal transduction?It is known from higher eukaryotes that signal transduction pathways, including proteins with reversible phosphorylation reactions, have a strong influence on isoform formation and are essential for developmental processes and environmental stress responses. Numerous genes, such as protein kinases, transcription factors, splicing regulators, and pathogen resistance genes, have been identified to regulate stress responses . The exp nucleus . It is t


Oryza sativa L.) is the main cereal crop in many Asian countries. The Asian rice planthoppers, Nilaparvata lugens (Stål) (brown planthopper), Sogatella furcifera (Horváth) (white-backed planthopper), and Laodelphax striatellus   (Hemiptera: Delphacidae), are the most economically important pests of rice. These three rice planthopper species often co-occur in the same paddy field. Traditionally, species identification of individuals of the three rice planthopper species has relied on morphological characters, but accurate discrimination of early-instar nymphs is very difficult, even for expert researchers. In this study, we developed a rapid one-step multiplex PCR assay using conserved and species-specific 5.8S-ITS2 rDNA gene primers for simultaneous identification of individuals of the three rice planthopper species. The multiplex PCR results showed that the three rice planthopper species could be identified accurately based on the length of the resultant amplicon, regardless of the individual developmental stage. Furthermore, we applied this assay for the first accurate quantification of early-instar nymphs of each rice planthopper species in paddy fields. Notably, we found that the species composition of early-instar nymphs cannot be extrapolated from that of adults. Thus, the multiplex PCR assay developed here facilitates detection of each rice planthopper species at the beginning of outbreaks in paddy fields.Rice ( Numerous insects migrate long distances between countries and continents , 2. SomeOryza sativa L.) is the staple food for more than half of the world’s population and more than 90% of the world’s rice is produced in Asia [Nilaparvata lugens (Stål) (brown planthopper), Sogatella furcifera (Horváth) (white-backed planthopper), and Laodelphax striatellus   (Hemiptera: Delphacidae), are the most economically important pests of rice [Rice  and ITS2R (5′-ATGCTTAAATTTAGGGGGTAGTC-3′) [® buffer (Mg2+ plus) , 4 μL of dNTPs (2.5 mM each), 2.5 μL of each primer (10 μM), 0.25 μL of Ex-Taq® DNA polymerase (TaKaRa Bio), and 2 μL of DNA template. The PCR cycling conditions consisted of an initial denaturation at 95° C for 3 min; followed by 40 cycles of denaturing at 94°C for 40 s, annealing at 56°C for 40 s, and extension at 72°C for 60 s; then a final extension at 72°C for 10 min. PCR products were gel-purified using a Wizard® SV Gel and PCR Clean-Up System  according to the manufacturer’s instructions, and sequenced by Eurofins Genomics  in both directions using the PCR primers. Consensus sequences were produced manually using BioEdit 7.0.4.1 [5.8S-ITS2 rDNA gene primers   . PCR was 7.0.4.1 . Primers 7.0.4.1 . Four 5.atellus) . The 5.8® buffer (Mg2+ plus) , 1.6 μL of dNTPs (2.5 mM each), 0.9 μL of the conserved forward primer , and 1 μL of DNA template. The PCR cycling conditions consisted of an initial denaturation at 95°C for 1 min; followed by 30 cycles of denaturing at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s; then a final extension at 72°C for 5 min. The PCR products were separated by 3% agarose gel  electrophoresis with 1× TAE buffer, stained with SAFELOOK™ Load-Green , and visualized by illumination with blue LED light.To validate the multiplex PCR primers, we used 10 wild-caught adults  randomly chosen from each of NL7–NL10, SF7–SF10, and LS7–LS10 , 10 laboN. lugens  and S. furcifera . We collected 3 adults  of N. muiri from Nagasaki, Japan, and 10 adults  of S. kolophon from Kumamoto, Japan. The multiplex PCR assay was performed as described above. To verify the integrity of the DNA used in this experiment, samples were amplified by PCR using the COI universal primers, Calvin (5′-GGRAARAAWGTTAARTTWACTCC-3′) [5′-TTGATTTTTTGGTCAYCCWGAAGT-3′) [® buffer (Mg2+ plus) , 1.6 μL of dNTPs (2.5 mM each), 1 μL of each primer (10 μM), 0.1 μL of Ex-Taq® DNA polymerase , and 1 μL of DNA template. The PCR cycling conditions consisted of 40 cycles of denaturing at 94°C for 1 min, annealing at 45°C for 1 min, and extension at 72°C for 1 min; then a final extension at 72°C for 10 min. The PCR products were analyzed by agarose gel electrophoresis as described above.As a preliminary analysis, we also applied the multiplex PCR assay to individuals of congeners of CTCC-3′)  and C1-JAAGT-3′) . PCR was® Spray; SDS Biotech, Tokyo, Japan) was held horizontally on one side of a rice hill at the bottom (about 2 cm above the paddy water), and the rice hill was beaten twice by hand to cause planthoppers to drop onto the sticky board. This procedure was repeated 20 times to collect planthoppers from 20 rice hills with one sticky board; three boards were used for each paddy field. The planthoppers captured on the boards were transported to the laboratory, and individual planthoppers were classified according to their developmental stage . For adults, individual planthoppers were morphologically identified to the species level according to Ref [To quantify the abundance of each rice planthopper species in paddy fields, we sampled planthoppers using a standard sticky board method (modified from Ref ) in two g to Ref . For nymN. lugens, n = 166; S. furcifera, n = 166; L. striatellus, n = 166)  .N. muiri and 10 adults of S. kolophon were tested using the multiplex PCR assay. The multiplex PCR primer set generated no clear band for any tested individual of these two planthopper species  were morphologically identified as three species . All collected nymph individuals  were early-instar nymphs, identified as one of three species of rice planthoppers by the multiplex PCR assay    . Moreove < 0.01) .N. lugens, S. furcifera, and L. striatellus). Our multiplex PCR assay enables robust and accurate species identification of Asian rice planthoppers, irrespective of developmental stage, sex, and collection date and locality . All collected individuals of early-instar nymphs were successfully identified as one of three species by the multiplex PCR assay . Thus, oS. furcifera in early-instar nymphs than in adults in paddy field A (S. furcifera [N. lugens in adults than in early-instar nymphs in paddy field B (N. lugens from mainland China to Japan in July 2019 [Nephotettix spp. (Hemiptera: Cicadellidae), that often co-occur in the same paddy field [It is generally assumed that the species composition of rice planthopper early-instar nymphs can be roughly estimated from that of adults in the same paddy field. Indeed, pest control advisors and farmers often evaluate the occurrence of early-instar nymphs by referring to the proportion of adults occurring at that time and use pest control strategies against the estimated most abundant planthopper species because it is difficult to discriminate early-instar nymphs of the three species . However field A  could beurcifera . The hig field B  could beuly 2019 . Hence, dy field , 41 and dy field . Our surN. lugens and S. furcifera , both of which are not pests of rice [In addition, our multiplex PCR assay was applied to congeners of  of rice , and no Uncontrollable outbreaks of Asian rice planthoppers led to a crisis in paddy ecosystems –18. The S1 FigNilaparvata muiri; SK, Sogatella kolophon.L, 100 bp DNA ladder; NM, (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


While a low vitamin D state has been associated with an increased risk of infection by SARS-CoV-2 in addition to an increased severity of COVID-19 disease, a causal role is not yet established. Here, we review the evidence relating to i) vitamin D and its role in SARS-CoV-2 infection and COVID-19 disease ii) the vitamin D status in the Irish adult population iii) the use of supplemental vitamin D to treat a deficient status and iv) the application of the Bradford-Hill causation criteria. We conclude that reverse causality probably makes a minimal contribution to the presence of low vitamin D states in the setting of COVID-19. Applying the Bradford-Hill criteria, however, the collective literature supports a causal association between low vitamin D status, SARS-CoV-2 infection, and severe COVID-19 . A biologically plausible rationale exists for these findings, given vitamin D’s role in immune regulation. The thresholds which define low, deficient, and replete vitamin D states vary according to the disease studied, underscoring the complexities for determining the goals for supplementation. All are currently unknown in the setting of COVID-19. The design of vitamin D randomised controlled trials is notoriously problematic and these trials commonly fail for a number of behavioural and methodological reasons. In Ireland, as in most other countries, low vitamin D status is common in older adults, adults in institutions, and with obesity, dark skin, low UVB exposure, diabetes and low socio-economic status. Physiological vitamin D levels for optimal immune function are considerably higher than those that can be achieved from food and sunlight exposure alone in Ireland. A window exists in which a significant number of adults could benefit from vitamin D supplementation, not least because of recent data demonstrating an association between vitamin D status and COVID-19. During the COVID pandemic, we believe that supplementation with 20-25ug (800–1000 IU)/day or more may be required for adults with apparently normal immune systems to improve immunity against SARS-CoV-2. We expect that higher monitored doses of 37.5–50 ug /day may be needed for vulnerable groups . Such doses are within the safe daily intakes cited by international advisory agencies. It reflects both dietary intake and cutaneous synthesis  and cutaneous synthesis and the sequential hydroxylation steps converting vitamin D to 25(OH)D and through to the final production of the active hormone 1.25(OH)2D  is schemynthesis  and is tynthesis . All majynthesis .Although the bulk of research on vitamin D physiology involves analysis of 25(OH)D and 1.25(OH)D, recent work has demonstrated other metabolic pathways that may be important for vitamin D function. These include pathways incorporating the hydroxylation enzyme CYP11A1. This enzyme catalyses the first step in the steroidogenic pathway, the conversion of cholesterol to pregnenolone. It is now recognized that substrates other than cholesterol, including vitamin D, can be hydroxylated by CYP11A1 in positions other than 25- and 1α- , 2015. TVitamin D deficiency is prevalent worldwide  and low i.e., not requiring mechanical ventilation) have 25(OH)D levels of less than 50 nmol/L. In Wuhan, China, a cross-sectional study of 335 COVID-19 patients and 560 controls with 25(OH)D measured before the SARS-CoV2 pandemic revealed that patients with COVID-19 had a higher prevalence of vitamin D deficiency (<30 nmol/L) on admission than that observed in the 2018–2019 control groups  even after adjusting for sex, BMI and presence of comorbidities (p = 0.014). Additionally, serum vitamin D levels were found to be inversely correlated with disease severity; 82% of patients with severe COVID-19 presented with vitamin D deficiency compared to 60% of non-severe COVID-19 patients (p = 0.0004). D levels . . The obs0.0004). . Moreove0.0004). . For exa reverse . This th reverse . Studies reverse . Thus, rp < 0.021; RR (age≥50) = 1.02,p < 0.064), non-white race  and being likely vitamin D deficient  compared to likely vitamin D sufficient. Predicted COVID-19 rates in the vitamin D deficient group were 21.6%  vs 12.2%  in the sufficient group (−1)  (p < 0.001) (p = 0.08) [mean 25(OH)D of 72 ± 54 nmol/L in infected subjects compared to 74 ± 45 nmol/L in the non -infected]. Conversely, in Sicily, when 50 patients infected with SARS CoV-2 were compared to 100 non-infected control patients , median serum levels of 25(OH)D were significantly lower  in patients than in controls (p = 0.016) (p = 0.037)  that were tested for COVID-19 of whom 499 had 25(OH)D levels measured in the year before testing, and determined that COVID-19 positivity was associated with increasing age  . Howevercontrols , a diffecontrols . In Engl= 0.016) . In Saud= 0.037) . Meltzer= 0.037) . This is= 0.037) . Thus a Radujkovic et al. studied the impact of vitamin D status on the severity of COVID-19 symptoms in 185 patients . Most (64%) had a serum 25(OH) D less than 50 nmol/L and inpatients in particular had significantly lower median levels of vitamin D than outpatients . Those with low vitamin D on admission had a higher risk of requiring invasive mechanical ventilation  and a higher mortality rate . In India, of 154 patients infected with SARS-CoV-2, 63 patients with severe symptoms had significantly lower serum vitamin D (mean = 36 nmol/L) at the time of admission compared to the remaining 91 asymptomatic infected patients (mean = 70 nmol/L) (p = 0.0001). Only two critical patients had normal levels of serum 25(OH)D3 D3 . In an A25(OH)D3 .I2 = 0.0%) and non-adjusted for confounders  and adjusted studies that used the Cox survival method  indicated a significant association of vitamin D deficiency with mortality, while in adjusted studies that used logistic regression no relation was observed   . The obs: 76.6%) .in vitro  (by 10–19%) and SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) (by 50–60%)  . These frophages . Vitaminrophages . The posrophages . A low vrophages . Severe  disease . Togethe disease ; immunol disease .post hoc analyses essentially discount the possibility that the more favourable outcomes of vitamin D-supplemented patients in the Cordoba study were attributable to chance or methodological issues such as imperfect blinding or uneven distribution of pre-existing comorbidities  is notoriously problematic and these trials commonly fail for a number of reasons . First, The observed associations between low vitamin D and SARS-CoV-2 are consistent with findings in other respiratory illnesses. Data from 21,000 subjects across eight observational studies indicate that those with a serum vitamin D level <50 nmol/L have a 64% increased risk of community-acquired pneumonia . As alluIreland is an island (situated between 51 and 56° North) with low levels of sunlight in wintertime, resulting in a high prevalence of low vitamin D status despite the presence of some voluntary food fortification. Serum 25(OH) D levels less than 50 nmol/L are seen in up to 50% of the Irish adult population in winter and 25% of the population during the summer . The oveIn a larger convenience study of 24,302 rural and urban patients from community (52 ± 16.4 years), outpatient (54.4 ± 17.9 years), inpatient (70.2 ± 17.5 years) and nursing home (81.5 ± 11.7 years) settings in the West of Ireland, over 50% of adults had serum 25(OH) D levels less than 50 nmol/L, rising to 67% of nursing home residents . VitaminIn another large inner city convenience study of GP referrals, of whom 17,525 were aged over 50 years, low vitamin D status (<50 nmol/L) was evident in 40% . Figure. In a st2  or D3  are widely available with and without prescription in Ireland. Vitamin D2 is manufactured using UV irradiation of ergosterol in yeast, and vitamin D3 is produced with irradiation of 7-dehydrocholesterol from lanolin and the chemical conversion of cholesterol (2) and D3 (25(OH)D3) respectively. Vitamin D3 increases serum 25(OH)D levels to a greater extent and maintains high levels longer than vitamin D2  for older age groups . In the However, in Ireland, the oral daily dose of vitamin D required to maintain serum levels above 50 nmol/L in 97.5% of healthy Irish adults throughout the year has been calculated to be 1120 IU  with an Natural dietary sources of vitamin D include oily fish such as salmon, tuna, mackerel, sardines, herring, cod liver oil, shiitake mushrooms and meat such as liver and beef. Wild fish have higher concentrations of vitamin D than farmed. Food sources fortified with vitamin D include fortified milk, yogurts, breakfast cereals and infant formula .. The 20p < 0.001) and achieved an increase in the prevalence of vitamin D supplement users from 11 to 41% (p < 0.001). In 2011, a mere 8 years post the introduction of this strategy, 91% of non-vitamin D supplement users who consumed fluid milk products, fat spreads, and fish based on Finnish nutrition recommendations reached 25(OH)D concentrations greater than 50 nmol/L. Based on the current voluntary fortification practices in Ireland, one would need to consume either four bowls of fortified cereal, or 6 eggs, or 4 tins of sardines each day to maintain an oral intake of 10 μg (400 IU). Since sunlight and food sources of vitamin D do not provide adequate vitamin D for older adults in Ireland, and current voluntary fortification practices, while helpful, are ineffective on their own in terms of achieving adequate intakes in the population, it is important that the population take vitamin D supplements of sufficient dose to help avoid a low vitamin D state.In selected European countries, low serum 25(OH)D concentrations appear to be associated with increased mortality from COVID-19 . MoreoveA low vitamin D state is often used to describe patients at risk of bone disease (25(OH)D < 50 nmol/L) rather than infection. It remains unknown what constitutes “low” in terms of a vitamin D state that protects against infection. Not only is it likely that the threshold for bone health is different to that for immune health, it seems likely that much higher serum vitamin D levels , daily supplementation doses of 800–1,000 IU/d and higher  or those whose response to oral dosing is attenuated . Furthermore, these doses are well within the consensus safe daily intake (tolerable upper limit) of 4000 IU/d defined by The recommended daily vitamin D intake  ranges from 400 to 600 IU, European Food Safety Authority  to 400–8We recommend that all adults ensure an intake of 800 to 1,000 units (20–25 μg) of vitamin D3 or more per day. People who are housebound or work indoors or have obesity or dark skin pigmentation may need a higher dose of vitamin D3 supplementation to replete their low vitamin D state , in order to ensure that optimal 25(OH)D blood levels for immune function are reached. The use of serum 25(OH)D levels is a reasonable guide on vitamin D status. We strongly recommend that the Irish Department of Health and Health Service Executive adopt a policy of recommending increased vitamin D supplementation for all age groups not only during the COVID-19 pandemic but also on an ongoing basis .i) Recommended Vitamin D intake 800–1000 IU (20–25 μg) per day, with higher monitored doses e.g., 1,500–2000 IU (37.5–50 μg) per day for vulnerable groups who have confirmed or likely low vitamin D status .ii) 50% of Irish adults in winter and 25% in summer have levels <50 nmol/L.iii) Further risk factors for deficiency include not taking supplements, smoking, physical inactivity, living alone, low socioeconomic status and diabetes mellitus.iv) Deficiency of Vitamin D has an association with severe COVID-19 - both SARS-CoV-2 infection and severity of Covid-19 disease are higher with insufficient blood levels.


P < 0.001) with right ventricular pacing (RVP) and was shortened to 120.9 ± 15.2 ms after LBBP (P < 0.001). The mean LBBP threshold was 0.7 ± 0.3 V at 0.4 ms at implant and remained stable during follow-up. The left ventricular ejection fraction (LVEF) increased from 36.3% ± 6.5% to 51.9% ± 13.0% (P < 0.001) with left ventricular end-systolic volume (LVESV) reduced from 180.1 ± 43.5 to 136.8 ± 36.7 ml (P < 0.001) during last follow-up. LBBP paced beyond the site of block, which results in a low pacing threshold with a high success rate in infranodal AVB patients. LBBP improved LV function with stable parameters over the 12 months, making it a reasonable alternative to cardiac resynchronization pacing via a coronary sinus lead in infranodal AVB and PICM patients.His bundle pacing (HBP) can reverse left ventricular (LV) remodeling in patients with right ventricular (RV) pacing-induced cardimyopathy (PICM) but may be unable to correct infranodal atrioventricular block (AVB). Left bundle branch pacing (LBBP) results in rapid LV activation and may be able to reliably pace beyond the site of AVB. Our study was conducted to assess the feasibility, safety, and outcomes of permanent LBBP in infranodal AVB and PICM patients. Patients with infranodal AVB and PICM who underwent LBBP for cardiac resynchronization therapy (CRT) were included. Clinical evaluation and echocardiographic and electrocardiographic assessments were recorded at baseline and follow-up. Permanent LBBP upgrade was successful in 19 of 20 patients with a median follow-up duration of 12 months. QRS duration (QRSd) increased from 139.3 ± 28.0 ms at baseline to 176.2 ± 21.4 ms ( Right ventricular pacing (RVP) leads to left ventricular (LV) dyssynchrony and may result in symptoms of heart failure (HF), a syndrome known as pacing-induced cardiomyopathy (PICM) , 2. PICMThe objective of our multicenter study was to assess the feasibility, safety, and clinical outcomes of LBBP in patients with infranodal AVB undergoing a device upgrade for PICM as a result of chronic RVP.This was a retrospective multicenter study including all consecutive patients undergoing LBBP pacing between December 2017 and June 2019 at three centers  meeting the inclusion criteria. Patients >18 years of age who met the following inclusion criteria were enrolled in this study: (1) PICM patients, which was defined as a >10% decrease in left ventricular ejection fraction (LVEF) after chronic RVP resulting in LVEF ≤ 50%. (2) The pacing percentage of RVP was >40%. (3) All patients were assessed for the site of conduction block. Then, the patients in whom infranodal AVB was confirmed, LBBP was attempted in order to pace beyond the site of block. Patients with other causes of LV dysfunction, including myocardial infarction, valvular heart disease (>15%), frequent ventricular premature depolarizations, and uncontrolled hypertension (>160/100 mmHg), were not defined to have PICM and were excluded  along with a 12-lead surface ECG GE CardioLab EP Recording System 2000; GE, Milwaukee, Wisconsin, USA. LabSystem PRO, Bard Electrophysiology, 196 Lowell, MA, USA were recorded. The techniques for HBP and LBBP were described in detail in our prior study , 18. BriInfranodal AVB is defined as the intra-Hisian and infra-Hisian block shown as a split His, His potential to ventricle interval (HV) prolongation, or HV dissociation. In patients without an underlying escape rhythm, the pacing rate was decreased to 30–35 bpm to assess for an escape rhythm. If patients had a ventricular escape rhythm due to sinus bradycardia, atrial pacing was used to help test intrinsic conduction to determine the site of block. A HBP lead was used to record the His potential to help assess the site of block.In patients with AVB, the left bundle branch (LBB) potential was recorded , proceedElectrical dyssynchrony was assessed by QRS width and axis. They were compared between native, RVP, and LBBP configurations. QRS duration (QRSd) was measured in 12 contemporary ECG leads. Paced QRSd was measured from the pacing stimulus to the end of QRS complex. Normal frontal QRS axis was defined as −30° to 90°, left axis deviation (LAD) as −90° to −30°, moderate right axis deviation (RAD) as 90°-180°, and superior RAD as 180°-270°. The precordial lead transition in which the precordial lead R-wave amplitude is equal to or greater than the S wave amplitude was recorded.Patients underwent regular follow-up at 3 and 6 months and annually post implantation. Functional status was assessed by the New York Heart Association (NYHA) classification. Device thresholds were checked and adjusted as needed to maximize battery longevity. Echocardiograms were performed as clinically indicated for follow-up. At follow-up visits, R-wave amplitude, capture threshold, pacing impedance, and the percentage of LBBP were collected. Standard echocardiographic indices including LVEF, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVESV), and valvular regurgitation were acquired by an experienced physician in accordance with the American Society of Echocardiography guidelines . Lead-ret-test if the data were normally distributed and with the Wilcoxon signed-rank test for non-parametric data. Paired categorical data  were compared with the Wilcoxon test. For echocardiographic LVEF, LVEDD, LVESV, and parameters  that were collected at baseline and later multiple different time points, univariate analysis of variance for repeated measures was used to assess the effects of LBBP. A P value ≤ 0.05 was considered statistically significant. Data analyses were performed using SPSS version 20.0 .Continuous variables were expressed as mean ± SD or median [interquartile range (IQR)]. Paired comparisons were made with a Student's Twenty PICM patients with confirmed infranodal AVB were referred for LBBP upgrade. One patient had failed LBBP lead fixation and was left with LVSP . Thus, 1P < 0.001) and shortened to 120.9 ± 15.2 ms with LBBP (P = 0.006). Pre-implant mean QRS axis of all patients was 32.63° (IQR: 0°-70.5°) and was 5.3° by RVP (IQR: −66.00°-80.5°) and kept stable at 63.84° after LBBP (IQR: 41.5°-73°). The percentage of patients with normal QRS axis was 84.2% (n = 16) on native and decreased to 26.3% (n = 5) on RVP (p = 0.0001) and was increased to 78.9% (n = 15) on LBBP (p = 0.001).Eleven patients had an intrinsic rhythm, with a narrow QRS in five, right bundle branch block (RBBB) in three, LBBB in two, and interventricular conduction delay (IVCD) in one . In patiEleven patients with native conduction had normal R-wave transitions between V3 and V4, while during RVP, 13/19 patients had an R/S transition from V5 to V6. During LBBP, 10 patients were noted to have a normal R/S transition between lead V3 and V4, while nine patients had an R/S transition between leads V1 and V2.p < 0.001, n = 18)   and the LVESV was reduced from 183.4 ± 44.7 to 137.2 ± 38.7 ml  (P < 0.001) with LVESV reduced from 180.1 ± 43.5 to 136.8 ± 36.7 ml (P < 0.001) during last follow-up, while an improvement in LVEF by ≥5% was observed in 17 patients (89.5%) and a super response was observed in five patients (26.3%).The echocardiographic measurements are summarized in  n = 18) , and the n = 18) . In pati n = 17) . The LVEp = 0.02). The number of patients with moderate-to-severe HF (NYHA III–IV) decreased from 73.7% (14/19) at baseline to 21.1% at 12 months (2/19) (P < 0.001). Of 18 patients who had diuretics before LBBP upgrade, nine patients were noted to have a reduction in diuretic dosage and another seven patients stopped diuretics completely. There was no difference in the number of patients taking angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist (ACEI/ARB) and β-blockers pre- and post-LBBP. In the year preceding LBBP upgrade, five patients (5/19) experienced at least one HFH, and only one patient (1/19) experienced HFH after LBBP (P = 0.027). One patient died 12 months after LBBP implantation due to ischemic stroke.During a median follow-up of 12 months, NYHA functional class was improved from 2.8 ± 0.6 to 2.1 ± 0.6 (As shown in The key findings from our study are as follows:LBBP could be performed safely in 95% (19/20) of patients with infranodal AVB who developed PICM.LBB pacing beyond the site of AVB had a low threshold and no loss of left bundle branch capture during the median 12-month follow-up.Favorable echocardiographic indices with improvement in EF, reduction in LV size, and improvement in NYHA class were observed in PICM patients by LBBP.LBBP was feasible in 95% of patients with infranodal AVB regardless of a narrow or wide QRS escape. LBBP captured the conduction system beyond the site of block, maintained LV synchrony, and reversed electrical dyssynchrony caused by RVP. LBBP improved LV function in PICM patients except for one patient with a native IVCD .After LBBP upgrade, 10 patients developed a normal QRS transition and nine patients demonstrated an R/S transition between leads V1 and V2. In our study, a normal QRS axis was maintained with LBBP in most cases and reversed PICM as we previously reported . LBBP deHBP could improve HF symptoms in LBBB as well as AF patients combined with atrial ventricular nodal (AVN) ablation , 25. ShaLBBP paced beyond the conduction block , 15, 26 In our study, no patients had lead dislodgment, perforation, or threshold increase during a median 12-month follow-up as indicated , 30. LBBIn a recent case report, LBBP reversed PICM from RVP . In our BVP seems to have a beneficial effect on left ventricular reverse remodeling, and the maximal effect of BVP is optimized by maximal fusion from LV lead pacing fusing with the intrinsic right bundle branch in patients with typical LBBB and QRSd longer than 150 ms . BVP couLBBP is a reliable physiological pacing strategy for AVB , 26, 27 This was a retrospective, multicenter, observational study with limited number of patients. The high success rates of LBBP achieved by experienced operators in our three centers need to be replicated in large pilot studies. Additionally, the success rates and the clinical outcomes of LBBP in this population must be interpreted with caution due to its retrospective study design. A dedicated prospective study is therefore warranted to answer this question. Another limitation of the study was the lack of a direct comparison to BVP as the standard treatment for PICM.This retrospective, multicenter, observational study demonstrates the beneficial effect and feasibility of LBBP in PICM patients with infranodal AVB. LBBP paced beyond the site of AV block with a low and stable capture threshold in 12 months as well as a high success rate of implantation in infranodal AVB patients.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the institutional review board. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.WH, GF, and XC conceived and designed the experiments. YY, SW, YS, XS, JZ, and BW analyzed and interpreted the data. YY, SW, XC, PS, and KE wrote or edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.