Background: Diabetes mellitus (DM) is associated with increased risk of sudden cardiac death (SCD), particularly in patients with heart failure with preserved ejection fraction (HFpEF). However, there are no known biomarkers in the population with DM and HFpEF to predict SCD risk.Objectives: This study was designed to test the hypothesis that osteopontin (OPN) and some proteins previously correlated with OPN, low-density lipoprotein receptor (LDLR), dynamin 2 (DNM2), fibronectin-1 (FN1), and 2-oxoglutarate dehydrogenase-like (OGDHL), are potential risk markers for SCD, and may reflect modifiable molecular pathways in patients with DM and HFpEF.Methods: Heart tissues were obtained at autopsy from 9 SCD victims with DM and HFpEF and 10 age and gender-matched accidental death control subjects from a Finnish SCD registry and analyzed for the expression of OPN and correlated proteins, including LDLR, DNM2, FN1, and OGDHL by immunohistochemistry.Results: We observed a significant upregulation in the expression of OPN, LDLR, and FN1, and a marked downregulation of DNM2 in heart tissues of SCD victims with DM and HFpEF as compared to control subjects (p < 0.01).Conclusions: The dysregulated protein expression of OPN, LDLR, FN1, and DNM2 in patients with DM and HFpEF who experienced SCD provides novel potential modifiable molecular pathways that may be implicated in the pathogenesis of SCD in these patients. Since secreted OPN and soluble LDLR can be measured in plasma, these results support the value of further prospective studies to assess the predictive value of these plasma biomarkers and to determine whether tuning expression levels of OPN and LDLR alters SCD risk in patients with DM and HFpEF. Heart failure with preserved ejection fraction (HFpEF) is the most common form of heart failure, affecting more than 3 million adults in the United States, and is represented by multiple subgroups or phenotypes . Type 2 A common cause of non-ischemic SCD in younger subjects is primary myocardial fibrosis , 5, whicOsteopontin (OPN) is a matricellular pro-fibrotic phosphorylated glycoprotein whose upregulation contributes to various pathological diseases such as cancers, chronic kidney disease, atherosclerosis, and adverse cardiac remodeling . In expeIn HF patients at high risk for SCD, plasma OPN and Galectin-3 levels were associated with sustained ventricular tachycardia and ventricular fibrillation . Plasma In this study, we investigated the expression levels of OPN and correlated proteins, dynamin2 (DNM2), low density lipoprotein (LDLR), 2-oxoglutarate dehydrogenase-like (OGDHL), and fibronectin-1 (FN1), in the hearts of SCD victims who had both DM and HFpEF. The correlation of the selected proteins with OPN is based on our previous findings that these proteins were positively or negatively correlated with OPN expression in the heart and/or kidney in a mouse model of HFpeF , 14. OurA death was classified as sudden if it was either a witnessed event within 6 h of the onset of symptoms or an unwitnessed death within 24 h when the subject was last seen alive in a normal state of health. The current criteria for SCD were chosen to cover as many subjects with SCD as possible, considering that many subjects with SCD would be found dead and would have been missed with the 1 h definition for SCD originally proposed by Hinkle and Thaler . All of Autopsied human heart samples from the left ventricular free wall from 9 SCD victims with DM and HFpEF and 10 previously healthy individuals who died as a result of an accident were obtained from a Finnish SCD registry as paraffin-embedded sections and analyzed for the expression of seven proteins by immunohistochemistry see .The study complies with the Declaration of Helsinki and was approved by the Ethics Committee of Northern Ostrobothnia Hospital District and the National Authority for Medicolegal Affairs . Consent from next of kin was waived by the Ethics Committee since according to the Finnish law, medicolegal autopsy does not require consent.The slides with paraffin-embedded heart sections were incubated for 45 min at 70 degrees, then dewaxed with two 5 min xylene washes and hydrated by 3 min graded ethanol washes of 100% (twice), 95, 80, and 70%, followed by two 4 min water immersions. Heat-mediated antigen retrieval was then done using 1X Citrate Antigen Retrieval Buffer (Ab93678) in a steamer for 75 min followed by permeabilization using 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 30 min, and then blocking with 10% donkey serum in 1% TBST for 45 min. The slides were then incubated with the following antibodies for 2 h at room temperature: osteopontin , low density lipoprotein receptor , dynamin 2 , fibronectin 1 , 2-oxoglutarate dehydrogenase-like , very low density lipoprotein receptor , and desmoplakin . Slides were then washed with PBS and staining was detected by incubation with various species-specific biotinylated secondary antibodies  at room temperature for 30 min, followed by DAB peroxidase (HRP) amplification . Primary and secondary antibody incubations were done in 10% donkey serum in 1% Tris-Buffered Saline, 0.1% Tween® 20 Detergent (TBST). Each single-antibody staining was repeated 3 times (on 3 different slides from the same paraffin block). No-primary control staining was performed with each of the secondary antibodies used.The stained slides were scanned at 20-X magnification using the Olympus VS120–L100 Virtual Slide Microscope . Images were quantified using ImageJ (NIH) by 2 independent examiners blinded to the groups. For each sample, 5 images (of 5 fields) were used for quantification and the average positive area normalized to the total stained area (as percentage) was used for statistical analysis.P-values were calculated using unpaired Student's t-tests and p < 0.05 was considered significant. Repeated symbols represent p-values of different orders of magnitude, i.e., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Similar significant differences were confirmed by two independent examiners.For all experiments, N refers to the number of individual subjects. All data are expressed as mean ± SEM. p < 0.05, p < 0.0001, We have previously shown that OPN and LDLR are highly expressed in the renal tubules of a mouse model of HFpEF, and that OPN knockcout reduced the pathologically high levels of LDLR and improved renal and cardiac function , 14. We p < 0.05, We have previously shown that OPN and DNM2 are highly expressed in the renal tubules of a mouse model of HFpEF, and that OPN knockcout reduced the pathologically high levels of DNM2 and improved renal and cardiac function , 14. Whep < 0.05, We have previously shown that in a mouse model of pressure overload, treatment with an OPN aptamer prevented cardiomyocyte hypertrophy and cardiac fibrosis, blocked OPN downstream signaling, and reduced expression of extracellular matrix proteins including FN1 . CardiacWe recently reported that OPN negatively regulates the expression of mitochondrial enzyme, OGDHL in the heart of a mouse model of HFpEF, and that OGDHL expression is downregulated in cardiac biopsies from patients with HFpEF and DM . We inveDiabetes Mellitus (DM) is a major public health problem affecting ~29.1 million Americans. DM is present in about 45% of patients with heart failure with preserved ejection fraction (HFpEF). Furthermore, SCD occurs in ~20% of HFpEF patients . Notablyin vivo. Therefore, we analyzed the protein expression levels of both OPN and its related molecules (previously shown to be correlated to OPN expression), as well as other proteins not previously correlated to OPN, to serve as negative controls.OPN is a matricellular protein that has been implicated in many inflammatory and profibrotic events, especially in the heart. In patients with severe aortic stenosis, elevated OPN levels correlated with increased rates of atrial arrhythmia, and an increased risk of death . In our 2-adrenergic receptor in cardiac cells  , 27. In Our team previously reported that the DSP gene, which codes for the structural protein Desmoplakin, harbors 2 variants associated with sudden cardiac death in subjects with primary myocardial fibrosis . FindingThe dysregulation of OPN, LDLR, DNM2, and FN1 protein levels in heart tissues . The patients/participants provided their written informed consent to participate in this study.KJ-W performed the experiments. MP, DR, KY, and LS analyzed the data and prepared the figures. MP, DR, KY, AL, AM, RG, LT, JG, RM, JJ, and LS interpreted the results of experiments. MP, DR, and KY drafted the manuscript. MP, DR, KY, KJ-W, AL, AM, RG, LT, JG, RM, JJ, and LS edited and revised the manuscript and approved the final version of 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.


In the negative ion mode, the two analytes and internal standard (IS) were monitored by multiple reaction monitoring (MRM), and the mass transition pairs were as follows: m/z 369.1 → 119.1 for parecoxib, m/z 313.0 → 118.0 for valdecoxib, and m/z 380.0 → 316.0 for celecoxib (IS). Six beagles were designed as a double cycle self-control experiment. In the first cycle, after intramuscular injection of parecoxib 1.33 mg/kg, 1.0 mL blood samples were collected at different times (group A). In the second cycle, the same six beagles were intravenously injected with 2 μg/kg dexmedetomidine for 7 days after one week of washing period. On day 7, after intravenous injection of 2 μg/kg dexmedetomidine for 0.5 hours, 6 beagle dogs were intramuscularly injected with 1.33 mg/kg parecoxib, and blood samples were collected at different time points (group A). The concentration of parecoxib and valdecoxib was detected by UPLC-MS/MS, and the main pharmacokinetic parameters were calculated by DAS 2.0 software. Under the experimental conditions, the method has a good linear relationship for both analytes. The interday and intraday precision was less than 8.07%; the accuracy values were from -1.20% to 2.76%. Cmax of parecoxib in group A and group B was 2148.59 ± 406.13 ng/mL and 2100.49 ± 356.94 ng/mL, t1/2 was 0.85 ± 0.36 h and 0.85 ± 0.36 h, and AUCt) of valdecoxib in group A were 37.79% and 36.19% higher than those in group B, respectively, and t1/2 was increased from 2.44 h to 2.91 h. Vz/F and CLz/F were correspondingly reduced, respectively. The developed UPLC-MS/MS method for simultaneous determination of parecoxib and valdecoxib in beagle plasma was specific, accurate, rapid, and suitable for the pharmacokinetics and drug-drug interactions of parecoxib and valdecoxib. Dexmedetomidine can inhibit the metabolism of valdecoxib in beagles and increase the exposure of valdecoxib, but it does not affect the pharmacokinetics of parecoxib.A sensitive and reliable ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of parecoxib and its metabolite valdecoxib in beagles. The effects of dexmedetomidine on the pharmacokinetics of parecoxib and valdecoxib in beagles were studied. The plasma was precipitated by acetonitrile, and the two analytes were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7  In clin effects . Evidenc effects .After intravenous administration, dexmedetomidine quickly distributes beyond total body water. Dexmedetomidine is metabolized by glucuronization and CYP2A6 hydroxylation and then excreted almost completely as a metabolite in urine , 6. BodyParecoxib  is a watIn particular, parecoxib was evaluated in pre- and postoperative treatments of gynaecological, ophthalmological, and colorectal surgery and intracranial and digestive tract tumour resection –13.The perioperative use of parecoxib can significantly improve the effectiveness of postoperative pain after transcatheter arterial chemoembolization (TACE), and parecoxib is effective and well tolerated in relieving pain caused by acute renal colic , 15.Dexmedetomidine and parecoxib are commonly used assistant anesthesia drugs in the clinic, and dexmedetomidine is often used in combination with methoxyphene, midazolam, dezocine, nonsteroidal anti-inflammatory drugs (NSAIDs), and other drugs for analgesia of anesthesia , 17. DexDexmedetomidine is a strong inhibitor of cytochrome P450 enzyme . ValdecoΩ) was prepared by a Millipore Milli-Q purification system.Parecoxib (purity > 98.0%), valdecoxib (purity > 98.0%), and celecoxib  were purchased from Sigma . Methanol and acetonitrile of LC-grade were provided by Tianjin Kermel Chemical Reagent Co., Ltd. Ultrapure water  was provided by Pharmacia and Upjohn company. Dexmedetomidine hydrochloride injection (LOT NO.: 180222BP) was obtained from Hengrui Medicine Co., Ltd.The equipment used in this study includes Waters Acquity UPLC instrument , XEVO TQD triple quadrupole mass spectrometer , ultrapure water equipment , and electronic analytical balance and vortex instrument.μm) by gradient elution with the mobile phase of 0.1% formic acid (A) and acetonitrile (B) at the flow rate of 0.4 mL/min. The gradient program was as follows: 0.00-0.50 min, 90% A; 0.50-1.00 min, 90-10% A; 1.00-2.00 min, 10% A; 2.00-2.10 min, 10-90% A; and 2.10-3.00 min, 90% A. The column temperature was set at 45°C, and the autosampler was conditioned at 4°C. An injection volume of 2 μL was applied for analysis.Parecoxib, valdecoxib, and IS were separated on an Acquity BEH C18 column .The XEVO TQ-S triple quadrupole mass spectrometer was used for mass spectrometric (MS) measurement with an electrospray ionization (ESI) interface in a negative ionization mode. Under the multiple reaction monitoring (MRM) conditions, quantification was achieved with transitions of Parecoxib, valdecoxib, and celecoxib were prepared at a respective concentration of 1.0 mg/mL by dissolving in methanol as the stock solutions. Various working solutions for the calibration curve and quality control (QC) were obtained in methanol by the gradient dilution of the stock solutions. The final concentrations of the calibration curves were covered by several points as follows: 5, 10, 50, 100, 500, 1000, 2000, and 4000 ng/mL for parecoxib and valdecoxib. As for QC samples, they were made in the same way at three concentration levels , and the concentrations of QC samples in plasma were 10, 500, and 3000 ng/mL for parecoxib and valdecoxib. The internal standard (IS) working solution (1000 ng/mL) was also prepared by diluting the stock solution of celecoxib with acetonitrile. All solutions were stored in a refrigerator at -20°C.μL of the IS working solution (1000 ng/mL in acetonitrile) was added to 100 μL of a plasma sample. The tubes were vortex mixed for 1.0 min. After centrifugation at 15,000 g for 15 min, the supernatant (2 μL) was injected into the UPLC-MS/MS system for analysis.Before analysis, frozen beagle plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 200 UPLC-MS/MS is a common method for the detection of biological samples, which has the advantages of rapidity, sensitivity, and accurateness. The UPLC-MS/MS method has been reported for the simultaneous determination of parecoxib and its metabolite valdecoxib , 23. In The specificity was assessed by comparing chromatograms of blank beagle plasma samples; blank plasma spiked with parecoxib, valdecoxib, and IS; and a beagle plasma sample 3.0 h after injection of parecoxib.χ2). The lower limit of quantification (LLOQ) was selected as the lowest concentration used in the calibration curve. The carryover test was performed by injecting a blank plasma sample injected with parecoxib and valdecoxib (4000 ng/mL) followed by a blank sample.The calibration curves were constructed and validated by analyzing spiked calibration samples for three days in a row. The linearity of the assay was assessed by analyzing the calibration curves using least-squares linear regression of the peak area ratios of the analytes to the IS versus the nominal concentration of the calibration standard with a weighed factor  in six replicates. On the same day, the intraday precision was calculated, and the interday precision was calculated by continuous measurement within 3 days. Precision was defined as the relative standard deviation  and accuracy as the relative error .Parecoxib and valdecoxib extraction recovery was compared by comparing the peak area of the conventional pretreated QC sample pretreatment with the peak area after extracting the corresponding blank plasma concentration (after extraction), and the matrix effect (ME) was evaluated by comparing the peak area of the analyte to water extraction after the sample and the corresponding replacement sample.Stability studies in biosamples were also conducted at three QC levels in several different storage conditions: room temperature for 12 h, -20°C for at least 4 weeks, after three freeze-thaw cycles (-20 to 25°C), and processed samples at 4°C in an autosampler tray for 12 h. The stock solution stability of parecoxib, valdecoxib, and IS was observed through six repeated tests. The stock solution portion stored at room temperature for 24 hours was compared with the remaining stock solution stored in a -20°C refrigerator to evaluate room temperature stability. The freezing stability was evaluated by comparing the newly prepared stock solution with the stock solution stored in a refrigerator at -20°C for three months.Six beagles  were obtained from Hubei Yizhicheng Biological Technology Co., Ltd, and the animal certificate was SCXK(hubei)2016-0020. The institutional approval number for the preclinical study of this experiment was 2019100013. The beagles were adapted to the new environment for 7 days in laboratory conditions. Necessary approval from the Institutional Animal Ethics Committee was obtained to carry out the experiments.Before the test, the beagles had free access to water but were fasted for 12 h. Six beagles were designed as a double cycle self-control experiment. Blood samples (1.0 mL) were collected from the forelimb cephalic vein or the small saphenous vein of the hindlimb and collected into heparinized tubes at 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 9, 12, and 24 h after intramuscular injection of parecoxib 1.33 mg/kg in the first cycle (group A). Blood samples were centrifuged for 10 min at 3000 × g, and the plasma was collected and kept frozen at -20°C until analysis, and the samples belonged to group A.μg/kg dexmedetomidine every morning in the second cycle (group B), with continuous injection for 7 days. On the seventh day, the six beagles were given 1.33 mg/kg parecoxib intravenously half an hour after intravenous injection of 2 μg/kg dexmedetomidine. The blood samples (1.0 mL) were collected into heparinized tubes at 0, 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 9, 12, and 24 h after intramuscular injection of parecoxib. Plasma was also separated and preserved, and the samples belonged to group B.After the one-week washout period, the same six beagles were injected intravenously slowly with 2 The above-developed UPLC-MS/MS method was used to simultaneously detect parecoxib and its active metabolite valdecoxib in the beagle plasma of group A and group B.The pharmacokinetic parameters of parecoxib and valdecoxib were calculated by the noncompartmental analysis using the DAS 2.0 software. All data were expressed as the mean ± standard deviation (SD).Under the above experiment, parecoxib, valdecoxib, and IS were well separated from endogenous substances. Representative chromatograms of a blank plasma sample (a), a blank plasma sample spiked with two analytes and IS (b), and a beagle sample (c) are shown in y = 1.5 × 10−3 x − 1.34 × 10−2 (r = 0.9993) and y = 1.8 × 10−3 x–3.82 × 10−2 (r = 0.9991), respectively, where y represents the peak area ratio and x represents the plasma concentration. The lower limit of quantification (LLOQ) of parecoxib and valdecoxib was 5 ng/mL. Carryover test results show that in the UPLC-MS/MS analysis, carryover does not affect the determination of parecoxib and valdecoxib.In the concentration range of 5–4000 ng/mL, the typical regression equations of parecoxib and valdecoxib were The recovery and ME results were investigated and are shown in All results for the stability samples tested are summarized in Tables The plasma concentration-time curve of parecoxib of group A and group B in six beagles after a single intramuscular injection of parecoxib is shown in t1/2, Cmax, Tmax, Vz/F, CLz/F, AUCt). Meanwhile, The DAS 2.0 program was used to calculate the main pharmacokinetic parameters of parecoxib and valdecoxib, including Cmax and AUC(0 − ∞) of valdecoxib in group A were 37.79% and 36.19% higher than those in group B, respectively, and t1/2 was increased from 2.44 h to 2.91 h. Vz/F and CLz/F were correspondingly reduced, respectively. It was suggested that dexmedetomidine can inhibit the metabolism of valdecoxib and increase the exposure of valdecoxib in beagles.Results show that after intramuscular injection of parecoxib, parecoxib was absorbed rapidly, and then, it was transformed into valdecoxib, an active metabolite. There was no significant change in the pharmacokinetics of parecoxib in groups A and B, it was suggested that dexmedetomidine does not affect the conversion of parecoxib. This study established a new UPLC-MS/MS method that can simultaneously detect the content of beagle dog plasma parecoxib and its metabolite valdecoxib. This method fully complies with bioassay standards, with high sensitivity and short analysis time (3 min). In the choice of plasma processing methods, we directly precipitate proteins with acetonitrile. The operation method is simple and fast, and the detection results are reliable. At the same time, the endogenous substances in the beagle dog plasma sample do not affect the determination of the analyte.When the pharmacokinetics (PKs) and pharmacodynamics (PDs) of a drug are changed by one or more drugs, drug-drug interactions (DDI) will occur . It is iNonsteroidal anti-inflammatory drugs, or NSAIDs, play antipyretic, analgesic, and anti-inflammatory effects by inhibiting COX and reducing the synthesis of prostaglandins. NSAIDs are available as oral tablets and solutions . MetabolParecoxib was rapidly and almost completely converted to valdecoxib. The elimination of valdecoxib was extensively carried out in a variety of ways in the liver, including cytochrome CYP3A4 and CYP2C9 enzyme metabolism and sulfonamide glucose hydroformylation (about 20%) .α2-adrenoceptor agonist with sedative, antianxiety, sympathetic, and analgesic effects [Dexmedetomidine is a highly selective  effects . Dexmede effects , 6. Dexm effects . The com effects . Dexmede effects .Cmax and AUC(0 − ∞) of valdecoxib in group A were 37.79% and 36.19% higher than those in group B, respectively, and t1/2 was increased from 2.44 h to 2.91 h. Vz/F and CLz/F were correspondingly reduced, respectively. It was suggested that dexmedetomidine can inhibit the metabolism of valdecoxib and increase the exposure of valdecoxib in beagles.The results of this study show that there was no significant change in the pharmacokinetics of parecoxib in groups A and B; it was suggested that dexmedetomidine does not affect the conversion of parecoxib in beagles. Although there are some differences in the pharmacokinetics of different species, the results of animal experiments can provide some reference for clinical medication . TherefoThe developed UPLC-MS/MS method for simultaneous determination of parecoxib and its metabolite valdecoxib in beagle plasma was specific, accurate, rapid, and suitable for the pharmacokinetics and drug-drug interactions of parecoxib and valdecoxib. Dexmedetomidine can inhibit the metabolism of valdecoxib in beagles and increase the exposure of valdecoxib, but it does not affect the pharmacokinetics of parecoxib.


These designs have been used successfully in agricultural and industrial research and in clinical trials for nearly a century without excessive levels of irreproducibility. They must also be used in pre-clinical research if the excessive level of irreproducibility is to be eliminated. A survey of 100 papers involving mice and rats was used to determine whether scientists had used the CR or RB designs. The papers were assigned to three categories “Design acceptable”, “Randomised to treatment groups”, so of doubtful validity, or “Room for improvement”. Only 32 ± 4.7% of the papers fell into the first group, although none of them actually named either the CR or RB design. If the current high level of irreproducibility is to be eliminated, it is essential that scientists engaged in pre-clinical research use “Completely randomised” (CR), “Randomised block” (RB), or one of the more specialised named experimental designs described in textbooks on the subject.Too many pre-clinical experiments are giving results which cannot be reproduced. This may be because the experiments are incorrectly designed. In “Completely randomized” (CR) and “Randomised block” (RB) experimental designs, both the  This leads to a waste of scientific resources with excessive numbers of laboratory animals being subjected to pain and distress3. There is a considerable body of literature on its possible causes7, but failure by scientists to use named experimental designs described in textbooks needs further discussion.Excessive numbers of randomised, controlled, pre-clinical experiments give results which can’t be reproducedrandomly intermingled within the research environment. Results can be statistically analysed using a one-way analysis of variance, with the usual assumptions of homogeneity of variances and normality of the residuals.Only two designs are suitable for widespread use in pre-clinical research: “Completely randomised” (CR) shown in Fig. RB design, the experiment is split up into a number of independent “blocks” each of which has a single subject assigned at random to each treatment. When there are only two treatments, this is known as a “matched pairs” design. The whole experiment consists of “N” such blocks where N is sample size. A two-way analysis of variance without interaction is used to analyse the results. The matched pairs design can also be analysed using a one-sample t-test.In the in-valid “Randomisation to treatment group” (RTTG) design, shown in Fig. order in which the experiment is done is not randomised. This is not valid because each treatment group will occupy a different micro-environment, the effects of which may be mistaken for treatment effects, leading to bias and irreproduciblity.Unfortunately, most authors appear to use the 8. Each variety was sown in several adjacent field plots, chosen at random, so that variation among plots growing the same and different crop varieties could be estimated. He used the analysis of variance, which he had invented in previous genetic studies, to statistically evaluate the results.Randomized controlled experiments have a long history of successful use in agricultural research. They were developed largely by R. A. Fisher in the 1920s as a way of detecting small but important differences in yield of agricultural crop varieties or following different fertilizer treatmentstwo sources of variation which need to be taken into account if true treatment differences are to be reliably detected. First, is the variation among the experimental subjects, due for example, to the number of grains in a given weight of seed, or to individual variation in a group of mice. Second is the variation caused during the course of the experiment by the research environment and in the assessment of the results. Both types of variation must be controlled if bias and irreproducibility are to be avoided.Fisher noted that in any experiment there are replication and randomisation so that plots receiving different varieties were randomly “intermingled” in the research environment.In most pre-clinical research the inter-individual variation can be minimised by careful selection of experimental subjects. But variation associated with the environment caused, for example, by cage location, lighting levels, noise, time of day and changes in the skill of investigators must also be considered. Fisher’s designs minimised bias by using uniform material and by 9“By randomization we mean that both the allocation of the experimental material, and the order in which the individual runs or trials of the experiment are to be performed, are randomly determined”.According to Montgomery10. The RB design is also convenient because individual blocks can be set up over a period of time to suit the investigator. Positive results will only be detected if the blocks give similar results, as assessed by statistical analysis12. Montgomery9,p 12 even suggests that blocking is one of the three basic principles of experimental design, along with “replication” and “randomisation”.The RB design often provides better control of both inter-individual and environmental variation. Subjects within a block can be matched and each block has a small environmental footprint, compared with the CR design. In one example this resulted in extra power equivalent to using about 40% more animals13, although they are not discussed here.Fisher and others invented a few other named designs including the “Split plot”, the “Latin square” and the “Cross-over” designs. These can also be used in pre-clinical research in appropriate situationsresearch environment may be the main source of inter-individual variation.In most pre-clinical experiments inter-individual variation can be minimised by choosing animals which are similar in age and/or weight. They will have been maintained in the same animal house and should be free of infectious disease. They may also be genetically identical if an inbred strain is used. So the 14. If two or more animals are housed in the same cage they will interact, this can increase physiological variation. Even external factors such as barometric pressure can affect the activity of mice15. Staff may also become more proficient at handling animals, applying treatments, doing autopsies and measuring results during the course of an experiment, leading to changes in the quality of data.Temporal variation due to circadian and other rhythms such as cage cleaning and feeding routines can affect the physiology and behaviour of the animals over short periods, as do physical factors such as cage location, lighting and noiseintermingled  the word “Statisticians involved in clinical trials sometimes write about “randomising patients to treatment groups”. Clearly, they are using the second definition as there are no physical groups in a clinical trial. But if scientists assign their animals to physical groups (“….things together”), they will be using the invalid “Randomisation to treatment group” (RTTG) design shown in Fig. A survey of published papers using mice or rats was used to assess the use of CR, RB, or other named experimental designs. PubMed Central is a collection of several million full-text pre-clinical scientific papers that can be searched for specific English words. A search for “Mouse” and “Experiment” retrieved 682,264 papers. The first fifty of these had been published between 2014 and 2020. They were not in any obvious identification number or date order. For example, the first ten papers had been published in 2017, 17, 19, 19, 19, 18, 15, 16, 19, and 18. And the first two digits of their identification numbers were 55, 55, 66, 65, 66, 59, 71, 61, 46 and 48. In order to introduce a random element to the selection, only papers with an even identification number were used.Each paper was searched for the words “random”, “experiment”, “statistical”, “matched” and other words necessary to understand how the experiments had been designed. Tables and figures were also inspected. The discipline and type of animals which had been used  was also noted. The aim was to assess the design of the experiments, not the quality of research.Most papers involved several experiments, but the designs were usually similar. All were assessed and re-assessed, blind to the previous scores, after an interval of approximately 2 weeks. The results in seventeen of the papers were discordant so they were reassessed.“Apparently well designed” . None of these papers mentioned either the CR or RB design by name, although a few of them appeared to have used one of these designs. For example, one stated: "All three genotypes were tested on the same day in randomized order by two investigators who were blind to the genotypes." This was scored as a CR design.“Room for improvement” . None of these papers used the word “random” with respect to the assignment of treatments to the animals, or the order in which the experiment was done, although it was sometimes used in other contexts. So these papers had not, apparently, used any named experimental design, so were susceptible to bias.“Randomised to group” . These papers stated that the subjects had been “Randomised to the treatment groups”. The most likely interpretation is that these were physical groups, so the experiments had used the statistically invalid RTTG design as shown in Fig. The results for mice and rats are summarised in Table Nineteen (38 ± 0.3%) of the rat papers were placed in the “Design acceptable” category. Those involving behaviour were of notably high statistical quality (and complexity). Three stated that they had used the “Matched pairs” design and one had used a RB design without naming it. None of them mentioned either the CR or RB designs.In eleven (22 ± 2.9%) papers, the rats were assigned to treatment groups. So it is unclear whether these papers had used the valid CR design or the in-valid RTTG design as discussed above for mice, although the latter seems to be more likely.The “Room for improvement” group consisted of 20 (40 ± 4.0%) of the papers. These had not used the word “random” with respect to the assignment of treatments to subjects or vice versa and there was no evidence that they had used the RB, CR or other recognised experimental designs.A similar search in Pubmed on “rat” and “experiment” found 483,490 papers. The first 50 of these with even identification numbers were published between 2015 and 2020. Four of them used mutant or genetically modified, the rest used wild-type rats. Twenty two of them involved experimental pathology, nineteen behaviour, seven physiology, one immunology and one pharmacology. Again, it was only the quality of the experimental design which was assessed, not the biological validity of results.p = 0.04). This was largely due to the high quality of the behaviour  studies in the rat.Results for both mice and rats are summarised in Table 17. Scientists wishing to build repeatability into their experiments could use the RB design, spreading the blocks over a period of time.Combining the two species, 32 ± 4.7% of the papers were judged to have been designed and randomised to an acceptable standard, although none of them stated that they had used either the CR or RB design. One mouse paper had used a “Latin square” design. Another had used a “Completely randomised” design without naming it, and a mouse paper noted that “All experiments were performed independently at least three times.” Such repetition can lead to tricky statistical problems if results are to be combined13. However, none of the papers in the above survey mentioned either the CR or the RB design by name, although these are the only designs suitable for general use.Names matter. People, places, species and scientific procedures have names which can be used to identify and describe a subject or a procedure. Experimental designs also have names; “Completely randomised”(CR), “Randomised block”(RB), “Latin square”, “Matched pairs” etc. These can be found in textbooks which describe the characteristics and uses of each designThe widespread use of the statistically in-valid RTTG design, which is not found in any reputable textbooks, may account for a substantial fraction of the observed irreproducibility. Organisations which support pre-clinical research and training should ensure that their literature and web sites have been peer reviewed by qualified statisticians and that they refer to named, statistically valid experimental designs.The RB and CR designs are quite versatile. They can be used for any number of treatments and sample sizes as well as for additional factors such as both sexes or several strains of animals, often without increasing the total numbers.clinical trials were supervised by statisticians who adapted the CR design for such work. But scientists doing pre-clinical research have received little statistical support, so it is not surprising that so many of their experiments are incorrectly designed. High levels of irreproducibility are unlikely to be found in pre-clinical research in the pharmaceutical industry because the “PSI”, , has about 800 members employed in the U.K.The first Irreproducibility is wasteful and expensive. The employment of more applied statisticians in Academia to assist the scientists doing pre-clinical research would be an excellent investment.


Finally, 580 papers were considered in detail. The number of published papers increased from 8 during the 1970s to 273 during the 2010s. These papers originated from 43 countries  and 240 institutions (145 universities and 95 other establishments), of which 35 produced ≥ 5 papers each. Most papers present original studies (n = 539) with a few reviews (n = 41). The original papers refer to dairy (n = 428), meat (n = 113), or wool (n = 1) production systems and present field (n = 329), laboratory (n = 163), or experimental (n = 67) work; the papers report aetiology (n = 146), risk factors (n = 100), pathogenesis (n = 92), diagnosis (n = 88), effects (n = 66), treatment (n = 50), control (n = 36), or descriptive epidemiology (n = 32) of the disease. Papers related to dairy production present more field and fewer experimental work than papers related to meat production; also, in papers describing work performed in dairy sheep, studies about aetiology, risk factors, and diagnosis of the disease predominate, whilst in papers performed in meat sheep, studies about aetiology, pathogenesis, and effects/diagnosis are reported more often. The papers were published in 175 scientific journals . On average, the papers received 16.8 total citations and 1.6 yearly citations (h-index = 47). Most papers were published in Scimago classification Q1 (n = 240) or Q2 (n = 230) journals and received 23.4 or 15.4 total citations, respectively. Reviews received more citations than original papers; among the latter, papers with work referring to dairy production received more yearly citations than papers referring to meat production; no differences in citations were seen according to type of work or mastitis aspect covered. Most citations were received by papers from France. Papers published in Journal of Dairy Science or Small Ruminant Research received the most citations. In total, there were 1558 individual authors of the papers, with 24 authors having co-authored > 10 papers each (max: 73 papers); on average, there were 5.2 co-authors per paper (min–max: 1–25). Average number of co-authors progressively increased from 2.1 in the 1970s to 6.3 in the 2010s, with original papers having a higher number of co-authors than reviews: 5.3 and 3.7, respectively. Papers from France had highers number of co-authors (7.9). The findings of this first ever scientometrics study into ovine mastitis indicate that the disease has not been studied as other sheep diseases and that future studies in it should be directed to its control.The present study is a scientometrics evaluation of refereed publications on bacterial mastitis in sheep; the objectives were the evaluation of the relevant papers and the presentation of quantitative characteristics regarding their scientific content and bibliometric details. The Web of Science platform was used with search terms: [ Scientometrics refers to the study of measuring and analysing scientific literature. Specific topics within this field refer to the measurement of the impact of research papers, the understanding of scientific citations, and the use of the results of such assessments in policy and management contexts . There aScientometrics and meta-science papers produce new data by using information in previously published research articles. Such articles present features and characteristics of scientific research, with the aim to produce a quantitative assessment of the initial research papers. They differ from review papers, which summarise the state of knowledge on a topic, without reporting new facts or carrying out new analyses. During assessment of the relevant literature, authors of reviews may discuss research papers and provide expert opinions by combining findings and ideas from the primary sources.In sheep, mammary infections can lead to bacterial mastitis (“mastitis”), mycoplasma mastitis , and lentiviral mammary infection. Among these, bacterial mastitis (“mastitis”) is the most important. It causes significant adverse financial consequences in sheep flocks and also reduces standards of welfare of sheep ,4. In reThe present study is a scientometrics evaluation of publications on bacterial mastitis (“mastitis”) in sheep during the last 50 years, from 1970 to 2019. Objectives of the study were the evaluation of the relevant publications and the presentation of quantitative characteristics regarding their scientific content and bibliometric details.www.webofknowledge.com; Clarivate Analytics, Philadelphia, USA) was used for the search of relevant publications. Only publications in this platform were assessed and included in the study. Search terms employed were: [mastitis OR *mammary infection*] AND [sheep OR ewe* OR ovine]. Timespan was set to ‘Custom year range (1970–2019)’. The following Web of Science collections were included in the search: (i) Science citation index expanded 1970–2019 (1970 being the earliest year for which relevant information is provided by the platform) and (ii) Emerging sources citation index 2015–2019.The Web of Science platform , ‘reviews’ (n = 76), ‘proceedings papers’ (n = 43), and ‘data papers’ (n = 1), for further assessment.Papilloma virus infection of teats, contagious ecthyma, staphylococcal dermatitis, sheep pox) with no reference or association to bacterial mastitis were also excluded.The abstracts of all these 1326 records were read and evaluated in the platform. Papers that did not deal with mastitis in sheep  were excluded. Further, papers related solely to contagious agalactia or the mammary aspects of lentiviral infections were also excluded. Moreover, papers referring solely to infections of the teats and the udder skin  and the scientific establishment(s) of only the first and the last authors were taken into account).Number of literature references included in the relevant list.Total number of citations received by the paper until the end of 2019.Number and names of all co-authors.▪Sheep production system to which the work referred: (i) dairy, (ii) meat, or (iii) wool; all systems that applied were assigned per paper.▪Type of study described in the paper: (i) experimental , (ii) field , or (iii) laboratory ; only one of these three types was assigned per paper.▪Mastitis aspect described in the paper: (i) aetiology, (ii) control, (iii) descriptive epidemiology, (iv) diagnosis, (v) effects, (vi) pathogenesis, (vii) risk factors, or (viii) treatment; up to two of these were assigned per paper.Type of paper: (i) original  or (ii) review . For original papers, the following details were also recorded.After the above abstract review, 580 papers were considered for detailed evaluation. All these were individually assessed. In each paper, the following details were recorded.p < 0.05.All data were entered into Microsoft Excel. Descriptive analysis was performed initially. The frequency of the various outcomes was evaluated in tables of cross-categorised frequency data by use of the Pearson chi-square test as appropriate. Comparisons between continuous data were performed by use of one-way analysis of variance. Correlations were performed as indicated and significance of the result was evaluated. Statistical significance was defined at All the 580 papers individually assessed are indexed in the Web of Science, fulfil the search criteria, and present work on ovine mastitis. A complete list of these papers with their details is in The number of papers published per decade in the topic increased progressively: 8 (1.4%) papers were published in the 1970s, 22 (3.8%) in the 1980s, 109 (18.8%) in the 1990s, 168 (28.9%) in the 2000s, and 273 (47.1%) in the 2010s .Papers originated from 43 different countries . Most pap < 0.001). Papers from Brazil, Greece, and Italy are the most recent ones , whilst papers from Australia and the United States of America are the oldest ones  (Differences in the year of publication are significant between the above 11 countries (ctively) .p < 0.0001). The establishments that produced more papers are the University of Thessaly , the University of Leon, the University of London , the Aristotle University of Thessaloniki, the National Institute of Agronomic Research France , the Complutense University of Madrid, the Veterinary University of Vienna , the Kimron Veterinary Institute Israel , the Commonwealth Scientific and Industrial Research Organisation Australia , and the University of Palermo .Papers originated from 240 different scientific establishments around the world, of which 35 published at least 5 papers each . Of thesMost papers presented original studies , and fewer ones presented reviews . Most original papers referred to dairy production , with fewer ones referring to meat  or wool (n = 1) production. Most original papers presented field work , and fewer ones presented laboratory  or experimental  work. Original studies reported mostly aetiology , risk factors , pathogenesis , or diagnosis  of the disease, whilst fewer papers existed on descriptive epidemiology , control , treatment , or effects  of the disease .p = 0.0036).There are clear differences in the type of work presented in papers according to the production system to which the study referred. Papers related to dairy production presented proportionately more field and fewer experimental work than papers related to meat production; for dairy production, the number of papers presenting field, laboratory, and experimental work were 272, 113, and 43, respectively , whilst respective figures for papers related to meat production sheep were 58, 31, and 24  (p = 0.044).There were also some differences in the mastitis aspect reported in the papers according to the production system. In papers performed in dairy sheep, studies about aetiology, risk factors, and diagnosis predominated , whilst in papers performed in meat sheep studies about aetiology, pathogenesis, and effects/diagnosis were reported more often  of the disease (p = 0.0065), in the production system being referenced (p < 0.0001), in the type of studies performed (p < 0.0001), and in the mastitis aspect presented (p < 0.0001). Similar differences were also recorded when only the 6, the 4, or the 3 countries with the most papers were included in the analysis   .Small Ruminant Research , Journal of Dairy Science , Veterinary Microbiology , Journal of Dairy Research , Pesquisa Veterinária Brasileira and The Veterinary Record , The Veterinary Journal , and Journal of Veterinary Medicine B  ; cumulatively, 245 papers (42.2%) were published in these 8 journals.In total, the 580 papers were published in 175 scientific journals. Journals in which over 10 papers were published are the following: p < 0.0001s). There was no further pattern of statistical association between these 8 journals and the 11 countries with the most publications (p > 0.95)  originated from 10 of the 11 countries with the most papers ; in contrast, if all articles are taken into account, a smaller proportion  originated from these 10 countries ( > 0.95) .p = 0.045) as well as in the production system being referenced, the type of studies performed, and the mastitis aspect presented . Similar significant differences were recorded also when the comparisons were made with the findings of only the 4 or the 3 journals with the most papers included in the analysis . Review papers included significantly more references than did original papers, 81.6 versus 29.6, respectively (p < 0.0001); moreover, papers describing laboratory work included more references than ones describing experimental work and field work: 32.2, 30.7, and 28.2, respectively (p = 0.0310), but no differences existed between papers according to mastitis aspect therein (p = 0.48) . Papers published since 2000 included significantly more references than those published earlier ( = 0.48) .h-index was 47 and 10i-index was 280. There was a clear correlation between the number of literature references in a paper and the number of citations received . When original and review papers were considered separately, a correlation was also evident between references and yearly citations , but not between references and total citations .The papers received (until the end of 2019) on average 16.8 ± 1.0 total citations and 1.6 ± 0.1 citations per year in the Web of Science platform. For all the papers, p < 0.0001).With regard to Scimago classification (year: 2019), most papers were published in Q1 (n = 240) or Q2 (n = 230) journals, with fewer ones in Q3 (n = 63) or Q4 (n = 22) journals or in journals outside this classification (n = 25). Papers in Q1 journals received more citations and more yearly citations than papers in Q2 or in Q3/Q4 or outside the classification journals: 23.4 ± 2.0 and 2.3 ± 0.2, 15.4 ± 1.5 and 1.4 ± 0.1, and 5.3 ± 0.5 and 0.5 ± 0.1, respectively (p < 0.0002). Most citations  were received by papers with an origin in France; these were published mostly (74.0%) in Q1 journals (p < 0.0005). Also, papers with origins from establishments in France  were the ones with most total citations and total yearly citations; these establishments also published papers mostly in Q1 journals (≥80%). However, establishments outside France  were the ones with most total cites , but fewer total cites . Papers with work referring to dairy production received significantly more yearly citations than did papers referring to meat production, although no differences were evident in the distribution according to journal classification (p = 0.0006), no significant differences in the citations received between papers of the three types were found (p > 0.59). With regard to mastitis aspect, differences were not significant in the classification of the journals in which papers were published (p = 0.067); also, differences in the average yearly citations received by papers did not differ according to the mastitis aspect (p = 0.407). Details are in On average, reviews received more total citations and total yearly citations per paper  than did original papers (15.4 ± 0.8 and 1.4 ± 0.1) . There were no correlations between the Scimago classifications of the journals and the citations received by papers published therein  (Papers published in arisons) .In total, there were 1559 individual authors of the papers; among these, 24 authors co-authored over 10 papers each (max: 73). Cumulatively, in the 580 papers, there were 2993 co-authors, i.e., on average 5.2 ± 0.1 co-authors per paper . Furthermore, there were 640 individual authors who were first or last authors in the papers; among these, 6 authors were first or last in over 10 papers each (max: 62). On average, the 24 authors with >10 papers each were first or last authors in 53.0 ± 6.1% of the papers in which they were co-authors .p < 0.0001). In 30 papers (5.2%), there was only one author.The average number of co-authors per paper progressively increased from 2.1 ± 0.4 and 2.6 ± 0.2 in the 1970s and 1980s, respectively, to 3.7 ± 0.2 and 4.8 ± 0.2 in the 1990s and 2000s, respectively, to 6.3 ± 0.2 in the 2010s (p < 0.0001); Papers from France, Italy, and Greece had the highest number of co-authors , whilst papers from Australia and the United Kingdom had the lowest (3.3 ± 0.3 and 3.2 ± 0.3). All the above 24 authors with the most papers worked in one of the 11 countries  and in one of the 35 establishments  from which most papers originated. Among them, 2 authors published papers with origins from two different countries and 4 authors published papers from two different establishments.Moreover, there was a clear difference in the number of authors in the papers from the various countries (p = 0.0008). In contrast, there was no difference in the number of co-authors among the papers according to study type (p = 0.15) or mastitis aspect (p = 0.54) therein.Original papers had a significantly higher number of co-authors than did reviews: 5.3 ± 0.1 versus 3.7 ± 0.3  but not with total citations per paper . When only papers with > 5.2 authors  were taken into account, correlation was seen for both parameters  .There was a clear correlation between number of co-authors and yearly citations per paper (ctively) . Papers Ovine mastitis is an important disease of sheep. The disease leads to significantly reduced production in affected animals and adversely affects their welfare standards.Mannheimia haemolytica). Minett [Probably the earliest reference to ovine mastitis was in a French language dictionary by the French veterinarian Hurtrel d’ Arboval in 1823  in the l. Minett  gave an . Minett  reviewedrespiratory infection* OR pneumonia] and 1670 records for [abortion] during the same year range as in the current study (for comparison: 1326 records found in the present study at the respective stage of the search). Also, there are no relevant studies to compare the present findings to those for mastitis in other species. Again, an initial stage search as above revealed 11,196 records for [cattle OR cow* OR bovine] and 1504 documents for [goat* OR doe* OR caprine]. These findings lend weight to the idea that mastitis in ewes has not been studied extensively.Despite its importance, mastitis in sheep has not been adequately studied, and there are facets of the disease that still have not been clarified. There are no scientometrics studies available in the international literature that would allow comparison of the mastitis literature to that about other diseases of sheep. An initial stage search revealed 2099 records  for [Nevertheless, the progressive increase of published papers indicates that this lack of scientific work has been identified by researchers, and relevant studies have been designed and performed. Possibly, the recognition by the European Food Safety Authority  of the sMost published papers originated from countries in the para-Mediterranean area. This was expected given that a significant number of sheep exist in these countries (approximately 15% of the world’s sheep population) . Sheep pAs sheep production in the para-Mediterranean area is oriented towards dairy production, one may indicate that the adverse effects of mastitis   become eThe findings indicate that some aspects of the disease have been covered extensively, e.g., aetiology, risk factors, and diagnosis. This is reflected in the presence in the international literature of papers that reviewed specific facets of the disease, e.g., ,14,15, oThe findings also indicate the accumulation of research on ovine mastitis in some establishments. This is a trend that is observed with many research topics. Hosting institutions and researchers accumulate expertise on a topic and capitalize upon it to further expand their work. The presence of large groups also seems to favour scientific output; among the authors with many papers on the subject, most originated from the same establishment, which indicates not only large groups but also long-established ones play a role in supporting the increased number of publications from those groups and of citations to those publications.Small Ruminant Research) or a thematic approach to dairy studies  rather than journals of general interest. This indicates the increased importance of topical scientific journals and the preference of researchers to publish in them; in that way, researchers address themselves to a more specialised audience with greater interest in their work and the ability to understand it.The journals in which the papers were mostly published are ones with a specific approach to sheep (In conclusion, this is the first ever scientometrics evaluation of mastitis in sheep. The findings indicate that the disease has not been studied as much as other diseases of sheep. Future studies should be directed to the control of the disease by using knowledge obtained from work into its other facets.


Direct inhibition of tumor necrosis factor-alpha (TNF-α) action is considered a promising way to prevent or treat TNF-α-associated diseases. The trimeric form of TNF-α binds to its receptor (TNFR) and activates the downstream signaling pathway. The interaction of TNF-α with molecular-grade dimethyl sulfoxide (DMSO) in an equal volumetric ratio renders TNF-α inert, in this state, TNF-α fails to activate TNFR. Here, we aimed to examine the inhibition of TNF-α function by various concentrations of DMSO. Its higher concentration led to stronger attenuation of TNF-α-induced cytokine secretion by fibroblasts, and of their death. We found that this inhibition was mediated by a perturbation in the formation of the functional TNF-α trimer. Molecular dynamics simulations revealed a transient interaction between DMSO molecules and the central hydrophobic cavity of the TNF-α homodimer, indicating that a brief interaction of DMSO with the TNF-α homodimer may disrupt the formation of the functional homotrimer. We also found that the sensitizing effect of actinomycin D on TNF-α-induced cell death depends upon the timing of these treatments and on the cell type. This study will help to select an appropriate concentration of DMSO as a working solvent for the screening of water-insoluble TNF-α inhibitors. Multiple proinflammatory cytokines are members of the tumor necrosis factor (TNF) superfamily and are involved in several physiological processes, including tumor prevention and host protection ,2. A sliTNF is a homo-trimeric cellular cytokine that is converted into a soluble form via proteolytic cleavage of the transmembrane form. Monomeric units of the bell-shaped trimeric form are held together via noncovalent interactions between them ,10. The Most drug-like molecules have a medium size and contain 10–50 atoms in addition to hydrogen. They mainly constitute interlinked aromatic cores along with various substituents composed of heteroatoms such as P, N, S, O, and X . Because of this structure, they are highly polarizable and conformationally flexible. This flexibility contributes to the reactivity, solvation, and formation of crystal polymorphs . The preHere, we aimed to explore the effects of different concentrations of DMSO on TNF-α-mediated signaling pathways at the molecular level. We determined the concentration has a minimal effect and is well suited for the screening of compounds. We also predicted the inhibitory mechanism of action of DMSO on TNF-α by in vitro assays and in silico simulations.v/v) for 1 h at room temperature. The mixture was then incubated with human dermal fibroblasts (HDFs) for 24 h, which converted the final concentration of DMSO to 0.0005%, 0.005%, 0.05%, 0.25%, and 0.5%, respectively. It was followed by collection of the supernatant and an ELISA to measure the secretion level of human interleukin-8 (hIL-8) and hIL-6. We observed a decrease in the secretion of hIL-8 (TNF-α is produced by various immune cells and activates nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs) via its receptors (TNFR1 and TNFR2), thereby leading to the production of multiple inflammatory cytokines ,39,40,41of hIL-8 A and hILof hIL-8 B with thof hIL-8 C.Along with cell proliferation, TNF-α causes inflammatory cell death by activating associated death signaling pathways . To evalMultiple signaling pathways, including NF-κB and MAPK, are activated by the stimulation of TNFR1 with its ligand, TNF-α . For the2O; After we confirmed the inhibitory impact of DMSO on the TNF-α signaling pathway, the DMSO mechanism of action was evaluated in a protein cross-linking interaction assay with subsequent Western blot analyses. A perturbation in the homotrimeric form of TNF-α interferes with its functionality and causes the inhibition of cytokine secretion and cell death ,49,50. TTo understand the possible mode of interaction between DMSO and TNF-α, we performed molecular dynamics (MD) simulations by placing different numbers of DMSO molecules inside the hydrophobic cavity of the TNF homodimer. The simulations were performed for 30 ns A–E, and While optimizing the TNF-α-induced cell death, we pretreated HDFs and L929 cells with act-D, followed by treatment with TNF-α at different time points . After overnight incubation, we quantitated the effect by calculating cell viability in the MTT assay and found a gradual decrease in TNF-α-induced cell death with the increasing time gap between the two treatments. This phenomenon was more prominent in HDFs A than inIn drug discovery, solubility has a key effect on bioassays, in vivo formulation, and intestinal absorption. The solubility of a molecule depends upon the solution conditions and the structure of the molecule. The conditions of the solution are influenced by cosolvents, pH, additives, time, temperature, or ionic strength. The structure of the molecule determines hydrogen bonding, lipophilicity, crystal energy, molecular volume, and ionizability. DMSO is a polar organic aprotic molecule that is capable of dissolving diverse drug-like compounds because of its amphipathic nature . Owing tTNF-α is a strong proinflammatory cytokine with multiple effects on different cell types and plays a major role in inflammatory diseases, such as rheumatoid arthritis ,58. It iTNF-α has been found to induce the production of multiple inflammatory cytokines ,39,40,41Activation of the TNF-α signaling pathway leads to either cell death or cell proliferation and survival . The tri2 at 37 °C . DMSO , rmTNF-α , rhTNF-α , act-D , and BS3  were purchased from the companies mentioned in the parenthesis.HDFs (American Type Culture Collection (ATCC), Manassas, VA, USA) and L929 cells  were grown in high-glucose DMEM supplemented with 10% of fetal bovine serum (FBS) , 1% of a penicillin/streptomycin solution, and 0.2% of Normocin solution . All the cells were incubated in a humidified atmosphere containing 5% CO4/well and 1.5 × 104/well, respectively, and grown overnight. After the completion of treatment, the medium was replaced with a 10% MTT solution (100 μL/well), and the cells were incubated for 3 h, followed by replacement of the solution and incubation with DMSO (100 μL/well) for 30 min. Finally, absorbance was measured at 540 nm on a spectrophotometer .An MTT assay (Sigma-Aldrich) was performed to measure cell viability. Briefly, HDFs and L929 cells were seeded in 96-well plates  at a density of 10HDFs and L929 cells were seeded at appropriate densities (mentioned above) in 96-well plates and grown overnight under suitable conditions. The next day, HDFs and L929 cells were pretreated with 1 and 0.1 µg/mL act-D, respectively, and incubated for 15 min. After that, a mixture of 10 ng/mL rhTNF-α with different concentrations of DMSO (0.1–100%) or 1 ng/mL rmTNF-α with different concentrations of DMSO (0.1–100%) (each preincubated for 1 h) was applied to the HDFs and L929 cells, respectively. The cells were incubated for 24 h post-treatment, and survival was measured by the MTT assay. Cell viability was calculated with reference to the no-treatment control. The obtained values were normalized to the act-D treatment group and were then used to calculate the death attenuation rate (%) using the following formula:The mammalian protein extraction reagent, M-PER , was used to isolate total protein from treated cells. The Bicinchoninic Acid (BCA) Assay Kit (Sigma-Aldrich) was utilized to measure the protein concentration. A Mini-PROTEAN Tetra Cell and Mini Trans-Blot Electrophoretic Transfer Cell System  were used for protein separation and transfer, respectively. The membranes were immunoblotted with primary antibodies, including those against phospho- (p-)p65, p65, p-JNK, JNK, ERK, p-p38, p38, IKB, , p-ERK, and β-actin  with gentle shaking at 4 °C overnight. The next day, membranes were rigorously washed with PBS containing 0.05% of Tween 20 and incubated at room temperature with a peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody for 2 h. The SuperSignal West Pico ECL solution  was applied to detect proteins, and visualization was performed on a ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories).4/well) were seeded and grown overnight in a 96-well plate (BD Biosciences). The cells were treated for 24 h with a mixture of rhTNF-α and different concentrations of DMSO (0.1–100%) (preincubated for 1 h). The secretion level of IL-8 was assessed with the Human IL-8 Uncoated ELISA Kit , while that of IL-6 was measured using the Human IL-6 ELISA MAX Deluxe Kit . The absorbance in the plate wells was read on a microplate spectrophotometer system (Molecular Devices).HDFs (101/10 volume of 1 M Tris-HCl (pH 7.5). Proteins in the samples were separated by SDS-PAGE and processed for Western blotting analysis with an anti-TNF-α antibody .Different concentrations of DMSO were incubated for 1 h at 37 °C with 100 ng of recombinant TNF-α, followed by cross-linking with 4.8 mM BS3  for 30 min at room temperature. Next, the reaction was stopped with a −1 nm−1. During position-restraint simulations, the temperature was equilibrated with the V-rescale scheme for 100 ps, and the pressure equilibration was performed via the Parrinello–Rahman algorithm at 1 bar for 100 ps. Next, a 30 ns production run was carried out without backbone restraints, and all bonds involving hydrogen atoms were constrained by a linear constraint solver algorithm. The trajectory data were saved every 10 ps, and the time stamp of 0.002 ps was set for the simulation. Data analysis and visualization were carried out using the VMD software [The topology of DMSO molecules was obtained from the PRODRG server . MD simusoftware  and built-test in Microsoft Excel 2016. All data are presented as mean ± SEM. p values < 0.05 were assumed to indicate statistical significance.All in vitro data analyses were performed by the two-tailed paired Student’s Drug-like compounds are usually dissolved in the polar organic solvent DMSO for the initial screening process. DMSO by itself can affect the target molecule. For example, TNF-α is an inflammatory cytokine that is involved in multiple diseases such as rheumatoid arthritis, Crohn’s disease, and psoriasis. The presence of DMSO as a solvent interrupts the functioning of TNF-α at the molecular level in terms of activation of associated signaling pathway components, induction of caspase-mediated cell death, and induction of secretion of ultimate inflammatory cytokines, such as IL-8 and IL-6. This inhibition is directly linked to the concentration of DMSO present in the solution. In some cells, sensitization by transcriptional arrest is necessary for the cell death pathway to prevail over cell survival/proliferative pathways. This sensitization process depends on the cell type and on the timing of treatments. According to our results, a proper time gap between act-D and TNF-α treatments, a suitable concentration of added DMSO, and/or a suitable alternative solvent for drug-like compounds must be considered for the screening of drug-like compounds.


Arthrobacter phage Scuttle was isolated by enrichment from a dry soil sample  on host Arthrobacter sp. ATCC 21022. The genome of this phage is 43,729 bp long, has a GC content of 61.1%, and has 61 annotated protein-coding genes. Arthrobacter phage Scuttle was isolated by enrichment from a dry soil sample  on host Arthrobacter sp. ATCC 21022. The genome of this phage is 43,729 bp long, has a GC content of 61.1%, and has 61 annotated protein-coding genes. Arthrobacter spp. are highly resistant soil bacteria that can withstand a multitude of environmental stressors, including starvation, heavy metal toxicity, free radicals, and harmful radiation , with coding sequences predicted by GeneMark v2.5p (Sequencing, assembly, and finishing of the genome were performed according to Russell . The phark v2.5p  and Glimrk v2.5p ; using Brk v2.5p , HHpred rk v2.5p , and manrk v2.5p , 61 protrk v2.5p  was usedrk v2.5p  or tRNAsrk v2.5p .Arthrobacter sp. ATCC 21022, at 61.1% versus 63.41%, respectively (Phage Scuttle contains 61 protein-coding genes mostly transcribed rightward, with 5 protein-coding genes located at the end of the genome which are transcribed leftward. The GC content of phage Scuttle is similar to that of its host ectively , 21. ScuMK814749 and SRA accession no. SRX8359893.Scuttle is available at GenBank with accession no.


Myotonic dystrophy type 1 (DM1) is the most common disease that can cause muscle weakness and atrophy among adults. Normal pressure hydrocephalus (NPH) is characterized by the triad of gait disturbance, cognitive impairment and urinary incontinence. The association between DM1 and NPH is extremely rare. We report a Chinese female patient with DM1 in association with NPH.The patient presented with a history of 3-year of walking instability and cognitive impairment. Her brain MRI showed ventriculomegaly with normal cerebrospinal fluid (CSF) pressure and the CSF tap-test was positive, which indicated the diagnosis of probable NPH. DM1 was confirmed by genetic testing.Four patients with DM1-NPH association were found before. The association between NPH and DM1 may not be just a coincidence, NPH may occur in DM1 later in life and it is vital to recognize the association as a shunt surgery may improve patients’ quality of life. Myotonic dystrophy type 1 (DM1) is the most common type of muscular dystrophy in adult. It is an autosome dominant disease which is associated with abnormal expansion of the repeated cytosine-thymine-guanine (CTG) in the 3′-untranslated regions in dystrophia myotonica-protein kinase (DMPK) gene on chromosome 19q13.3 . While tNormal pressure hydrocephalus (NPH) is a neurological disease characterized by the triad of gait disturbance, cognitive impairment and urinary incontinence, presenting ventriculomegaly and normal cerebrospinal fluid (CSF) pressure. It was initially described in 1965 and can be treated via ventriculoperitoneal shunt surgery . The preThe coexistence of myotonic dystrophy (DM) and NPH has been reported in four patients so far Table  14–16].–16.14–16A 65-year-old Chinese woman with 1 year of grade school education came to the Department of Neurology, First Affiliated Hospital of Zhejiang University in November 2018. Her complaints included a 3-year history of walking instability which was characterized by slow walking, lower foot height, hypsokinesis, poor balance, reduced stride length, broad-based gait, and difficult starting/turning. Furthermore, she was found to have memory loss and was slow to respond. The symptoms had gradually worsened, and she was dependent on family members to help her walk. Twenty days before presenting to our clinic, she suffered thoracic vertebral compression fracture (T11–12) due to a fall at home and required surgery. The patient had no medical history of hypertension, diabetes, cardiac diseases. She had no family history of similar diseases. Her neurological examination revealed a severe gait disturbance and increased muscular tone of limbs. Marked muscle wasting was seen in the temporalis. The Medical Research Council power was 4/5 on the flexor and extensor of four limbs , cholecystolithiasis in abdominal ultrasound, bilateral multiple thyroid nodules in the thyroid ultrasound, and chronic inflammatory foci in the lower lobes of both lungs in lung CT. Furthermore, brain MRI revealed ventriculomegaly (Evans index =0.34)  accompanying with normal CSF pressure and ventricular enlargement. These observations triggered a probably case of NPH  and cereCurrently, the definite diagnosis of DM1 is made by DNA analysis. By contrast, in the past, non-molecular testing has been widely adopted which plays no role now  due to iGenetic testing showed that the patient had 100 CTG repeats in the DMPK gene. Consistent with a clinical presentation of DM1, the patient exhibited muscular weakness, muscle wasting of temporal and distal limb muscles. Additionally, myogenic damage and excessive myotonic discharge were noted on the EMG. For the last 3 years, she had experienced progressive gait disturbance and cognitive decline. Brain MRI showed enlarged ventricles with an Evans index of 0.34 and CSF pressure was normal. Ventricular enlargement in the presence of normal CSF pressure pointed to NPH , which wDM1 brains show general atrophy and widespread gray matter volume reductions . It is wCerebral ventriculomegaly is commonly reported in DM1 patients –37 and aThe mechanism of NPH may be the obliteration of arachnoid villi , leading to the disturbances of CSF reabsorption, leading to disrupted CSF reabsorption and subsequent ventricular enlargement , 49. TheTwo of the four patients with DM1-NPH association received shunt surgery Table , also inOverall, for elderly patients with DM1 presenting symptoms of gait disturbance or urinary incontinence, it is vital to consider the association of NPH. Once DM1-NPH association is considered, shunt surgery should be suggested and the patient should be transferred to neurosurgery department. In this way, it could improve the patients’ quality of life.


Both isomers are characterized by metal–metal bonding with a high bond order; the orbital occupations point to quadruple bonding. Irradiation with ultraviolet light induces the transformation of linear HVVH to folded V2(μ‐H)2, whereas irradiation with visible light initiates the reverse reaction.Dinuclear compounds of early transition metals with a high metal–metal bond order are of fundamental interest due to their intriguing bonding situation and of practical interest because of their potential involvement in catalytic processes. In this work, two isomers of V Into the fold: Linear HVVH and folded V2(μ‐H)2 have been generated in solid Ne and were detected by infrared spectroscopy. Both molecules are characterized by multiple metal–metal bonding with a high bond order. A larger number of bare transition‐metal dimers has been generated in noble‐gas matrices,2 reacts with dinitrogen by formation of cyclic Ti2(μ‐N)2, breaking even the strong triple bond, whereas Ti atoms do not show a corresponding reaction.2+, which was shown to be able to activate ammonia in the gas phase (Pt2++NH3→Pt2NH++H2), whereas Pt+, Pt3+, and Pt4+ were not.2 has been investigated in matrices.2H2. The late‐transition‐metal compounds Rh2H22H22H2,2)2)An obvious way to generate HMMH molecules in inert‐gas matrices is the reaction of matrix‐isolated bare metal dimers with H2H2 molecules. So far, the reaction of V2 with H2 has not been reported, only V atoms have been reacted with dihydrogen. Thermally evaporated V atoms in Kr and Ar matrices have been found to react with H2 only upon irradiation (320 nm<λ<380 nm) by formation of VH2, whereas V atoms in their electronic ground state do not react with H2.2 to give VH2 in Ne and Ar matrices also without specific irradiation.In this work, we report on the generation and characterization of matrix‐isolated V2 has been studied,2+ reacts in the gas phase with D2 to yield V2D+.2+ with CO2 to yield V2O2+ has been investigated, and the surprising observation has been made that the intermediate V2O+ reacts considerably faster than V2+.Also, for some transition‐metal‐cluster cations, among them the dimer cations, the reactivity with respect to D2 reacts with H2 without a significant reaction barrier to linear HVVH under preservation of the spin multiplicity and to the V2(μ‐H)2 isomer with a nonplanar, folded structure  calculations based on complete‐active‐space self‐consistent‐field orbitals. Vibrational frequencies are calculated by density‐functional calculations.−1, and furthermore absorptions in the region between 331 and 346 cm−1 that belong to the same compound , as well as a band in the low‐frequency regime at 343.4 cm−1 with a shoulder at 342.5 cm−1 at the expense of the broader bands. Upon irradiation with visible light, the broader bands re‐appear with a slightly changed intensity pattern; there is more intensity at around 1580 cm−1 with two maxima at 1581.4 and 1580.2 cm−1, and in the low‐frequency region, there are two pronounced absorptions at 342.4 and 340.3 cm−1. Upon renewed annealing to 10 K, the sharper bands that appeared in the first annealing again recover intensity at the expense of the broader bands. Upon irradiation with ultraviolet (UV) light, all those bands are extinguished, and upon further annealing to 10 K, the sharp bands only very weakly re‐appear.The IR spectra recorded after co‐depositing vanadium atoms and a mixture of Ne and dihydrogen show broader absorptions at around 1580, 1571, 1569, and 1564 cm−1 , but neither directly after deposition nor directly after irradiation with visible light. These absorptions are found at 1425.1, 1405.3, 917.2, and 565.4 cm2 or HD. For the first set of absorptions, there are D2 counterparts of the four sharper signals that appeared after annealing to 10 K at 1132.6, 1131.9, 1131.8, and 1131.5(sh) cm−1, and counterparts in the low‐frequency regime at 247.0 and 246.2(sh) cm−1 , 1137.8, and 1137.4(sh) cm−1 near the absorptions found with D2. In the low‐frequency region, the HD counterparts are observed at 276.7 and 275.9(sh) cm−1. Furthermore, using HD, there appears another weaker band at 442.8 cm−1 not found in the experiments with H2 or D2.For all those bands, corresponding counterparts are observed using D2 counterparts are found at 1035.7, 1021.4, 678.0, and 399.3 cm−1  in solid NeFor the second set of absorptions, the D2(μ‐H)2 molecule with bridging H atoms. The assignment is based on the number of bands observed, the positions of the bands, and their isotopic shifts, and it is confirmed by computational results .The first set of bands is assigned to the linear HVVH molecule, the second set of bands is assigned to the folded V2, there is one band in the V−H stretching region at 1570.7 cm−1.  should be observable in infrared spectra, and indeed, another mode is observed at 343.4 cm−1, the ungerade combination of the bending motions. When using HD, due to symmetry reduction, yet another mode is observed at 442.8 cm−1, the formerly gerade combination (πg) of the bending vibrations. The density‐functional results nicely confirm the assignment (see below).Within the first set of bands, when using H2, there are two signals at 1425.1 and 1405.3 cm−1 in the region of the stretching vibrations. They have lower wavenumbers than the signals assigned to HVVH, consistent with H in a bridging configuration. The isotopic shifts to 1035.7 and 1021.4 cm−1 when using D2 confirm that these bands are related to the motion of H atoms. The observation of two bands in the V−H stretching region already with the homonuclear reactants points to a non‐symmetric, likely non‐planar, structure. For folded Cv2‐symmetric V2(μ‐H)2, five out of six modes  should, in principle, be observable in infrared spectra. Two additional modes are observed at 917.2 and 565.4 cm−1, the shift of the H atoms along the direction of the V−V axis at 917.2 cm−1, the folding motion at 565.4 cm−1. Thus, out of the five in principle observable modes, four are found. The density‐functional calculations confirm the assignment to folded Cv2 V2(μ‐H)2 and indicate that the other mode which, in principle, could be observed, is very weak, a mode dominated by the V−V stretching motion.Within the second set of bands, when using H2H2 a folded V2(μ‐H)2 isomer with a Cv2‐symmetric structure, and, at an energy higher by about 0.5 eV, a linear HVVH isomer with Dh∞ symmetry  at the structure of the Cv21A1 ground term are shown in Figure 3B2 and a 1B2 term at 0.79 and 0.89 eV. The lowest‐lying term of the linear HVVH isomer is, according to the MRCI calculations, a 3Σg− term at an energy of 0.48 eV relative to the 1A1 state. The V−V distance amounts to 170.7 pm and the V−H distance to 170.5 pm. The same term is also obtained by the density‐functional calculations at a relative energy of 0.56 eV, in good agreement with the MRCI result. The values for the V−V and V−H distances of 171.1 and 171.6 pm obtained by the density‐functional calculations are again close to the MRCI values and confirm the qualitatively correct picture of the density‐functional calculations also in the case of the linear isomer. The relative energies of the lower‐lying terms at the structure of the 3Σg− term are also shown in Figure 3Σg− term, there are a 1Γg and a 1Σg+ term, respectively. Further singlet and triplet terms follow at energies higher than 1.21 eV.According to the MRCI calculations, the electronic ground state of the folded 2(μ‐H)2. All vibrational modes for which the calculations indicate transitions with substantial intensity have been observed with wavenumbers not too far from the calculated values. For the linear isomer, the wavenumbers of the allowed transitions are calculated to be 1613 and 322 cm−1 2 isomer, the occupation of the valence‐space natural orbitals of the MRCI wave function, (8a1)1.97(3b2)1.96(9a1)1.85(10a1)1.81(4b2)1.77 (11a1)1.70(8b1)0.28(3a2)0.21(9b1)0.18(10b1)0.11(12a1)0.04(4a2)0.03 (5b2)0.02(11b1)0.01, clearly shows the multiconfigurational character. The leading configuration in the expansion of the wave function is (8a1)2(3b2)2(9a1)2(10a1)2(4b2)2(11a1)2 with a coefficient of 0.77. The first two orbitals are orbitals with a leading contribution from the H 1s orbitals. They are V−H bonding orbitals, the first one mainly involving the V 4s orbitals, the second one involving the 3d orbitals. The following eight orbitals are essentially 3d orbitals pointing to a substantial amount of 3d–3d bonding. Four of them are bonding orbitals and four of them are the corresponding antibonding orbitals. From the occupation numbers of the natural orbitals, a bond order of 3.17 is obtained. To confirm this consideration, a natural‐bond orbital analysis has also been performed. For the bridging hydrogen atoms, it yields two three‐center H−V2 bonding orbitals occupied with 1.96 electrons each and corresponding antibonding orbitals occupied with 0.03 electrons each. For the vanadium–vanadium interaction, it yields four V−V bonding orbitals occupied with 1.85, 1.81, 1.77, and 1.70 electrons, and corresponding antibonding orbitals occupied with 0.25, 0.21, 0.19 and 0.10 electrons. These values amount to 7.13 electrons in bonding orbitals and 0.75 electrons in antibonding orbitals, yielding a value of 3.19 for the bond order of the V−V bond, close to the value obtained by consideration of just the natural orbitals. Thus, the value of larger than three is indicative of the presence of fourfold bonding in V2(μ‐H)2.For the 3Σg− ground state of the linear HVVH isomer, the occupation of the natural orbitals for the MRCI wave function is (6σg+)1.97(6σu+)1.96(7σg+)1.89(3πu)1.86(3πu)1.86(1δg)0.90 (1δg)0.90(3πg)0.12(3πg)0.12(1δu)0.11(1δu)0.11(7σu+)0.09(8σg+)0.04 (8σu+)0.02. The leading configuration is (6σg+)2(6σu+)2(7σg+)2 (3πu)2(3πu)2(1δg)1(1δg)1 with a coefficient of 0.83. The first two orbitals are V−H bonding orbitals with the main contributions from H 1s and V 4s orbitals; the remaining ones are V 3d orbitals. Hence, the σ, π, and δ orbitals contribute to the V−V bonding. From the occupation numbers of the natural orbitals, a bond order of 3.43 is obtained, still somewhat larger than for the isomer with the bridging hydrogen atoms. The triplet ground state results from the occupation of the two δ orbitals with two electrons. An NBO analysis as well as the Mayer bond order based on the density‐functional results confirm the conclusions from the evaluation of the MRCI wave function (see the Supporting Information).For the 2(μ‐H)2 and the fragmentation of HVVH, additional density‐functional calculations were performed on V2(3Σg−), VH(5Δ), and H2. According to the calculations, the formation of V2(μ‐H)2 via V2+H2→V2(μ‐H)2 is exotherm by 1.18 eV. Hence, the formation of the linear HVVH isomer is still exotherm by 0.62 eV. The 5Δ ground state for VH is in line with previous calculations.3Σg−)→2 VH(5Δ) amounts to 2.77 eV. This value is similar to the experimental value of 2.75 eV for the dissociation energy of V22,To obtain an estimate for the reaction energy of the formation of V2(μ‐H)2, clear, sharp signals are observed only after annealing (not after deposition or irradiation), pointing to the population of a few (well‐defined) matrix sites. For HVVH, the broader signals found after deposition or irradiation are clearly apparent 2. Nevertheless, upon close inspection of the spectra after deposition or irradiation, also for V2(μ‐H)2, broad absorption features somewhat red‐shifted to sharper signals can be perceived 2 is also present in the matrices after annealing or irradiation, but that the signals are too broad to be clearly seen. Notice that the signals of V2(μ‐H)2 are, in general, weaker than those of HVVH: The strongest signal of V2(μ‐H)2 has only about one quarter of the intensity of the strongest signal of HVVH. Thus, the detection of broad signals is expected to be more difficult for V2(μ‐H)2.For both HVVH and Vd Figure . Additio2 reacts with V2 by formation of both a linear HVVH isomer and a folded Cv2 V2(μ‐H)2 isomer. Thus, it is likely that the reaction proceeds without or with only a small barrier. The folded isomer is the more stable one, nevertheless, the linear isomer is formed in substantial amount. This observation is consistent with the ground‐state properties of folded V2(μ‐H)2 and linear HVVH, as revealed by the calculations. HVVH has a 3Σg− ground state, the same as the diatomic V2. Thus, the formation of HVVH from V2 and H2 is a spin‐allowed process, whereas the formation of the folded V2(μ‐H)2 isomer, which has a 1A1 ground state, is spin‐forbidden although energetically favorable. Irradiation with UV light leads to the destruction of HVVH and almost exclusive formation of the more stable V2(μ‐H)2. Obviously, by irradiation with UV light, higher‐lying excited states are populated that allow an easy isomerization to the folded V2(μ‐H)2, for example, by change of the spin state due to spin–orbit coupling and subsequent isomerization or vice versa.The experiments show that H2(μ‐H)2 decrease, whereas those of HVVH increase, and upon irradiation with UV light, the signals of HVVH almost entirely vanish, whereas those of V2(μ‐H)2 markedly increase. The experiments indicate the conversion of linear HVVH to non‐planar V2(μ‐H)2 by irradiation with UV light and the conversion of V2(μ‐H)2 to HVVH by irradiation with visible light 2, whereas ground‐state Ga atoms do not react with H2. (GaH2 is formed only upon photoexcitation.)2(μ‐H)2 features an interesting photochemistry. Irradiation (λ=546 nm) of Ga2(μ‐H)2 leads to the formation of HGaGaH and H2GaGa, and upon irradiation of these products using other wavelengths (λ=365 nm or λ>700 nm), cyclic Ga2(μ‐H)2 is reformed. Furthermore, the present results for V2H2 are similar to the findings of a theoretical  study on Ti2H2.The chemistry of V2(3Σg−) of 180.4 pm,2, (6σg+)2(7σg+)2(3πu)2(3πu)2(1δg)1(1δg)1, even points to a quintuple bond . An explanation of this contradictory observation is likely the following: due to the formation of the V−H bonds, the 4s contribution to the electron density at the V atoms and in the V−V bonding region decreases, and therefore also the shielding of the V nuclear charge, and the V2 core has partial V22+ character leading to a contraction of the V−V bonding orbitals. Although for both V2(μ‐H)2 and HVVH, the results point to quadruple bonding, the V−V bond distance in V2(μ‐H)2 is longer by 13.7 pm than in HVVH. The values for the V−V bond order are in qualitative agreement with this observation: the values point to quadruple bonding for both isomers, but the value for HVVH it is still somewhat larger than for V2(μ‐H)2. However, the bonding situation differs in some respects. In V2(μ‐H)2, there are bridging H atoms, and hence, there is associated electron density close to the V−V axis. Furthermore, HVVH has a triplet ground state. Therefore, in its leading configuration, two of the five orbitals that contribute to the bonding are occupied by one electron only, whereas in V2(μ‐H)2, there are four V−V bonding orbitals occupied by pairs of electrons. Thus, in HVVH, for the two unpaired electrons, there is the possibility to keep a larger distance. It is likely that both factors influence the final bonding situation.The V−V bond distance of 170.7 pm for HVVH is surprisingly short, even shorter than that of V2(μ‐H)2 and HVVH, respectively, are still shorter than the values in the paddlewheel complexes V2(DPhF)4 with a formal triple bond (197.8/197.9 pm)2(DPhF)4] with a formal bond order of 3.5 (192.9 pm),24+ and V23+ cores, and far shorter than the value of 246.0 pm in a divanadium(IV) complex with a V−V single bond,2H2. HVVH and V2(μ‐H)2 are further examples of bimetallic metal hydride compounds with a high metal–metal bond order, like HCrCrH, HMoMoH, and HWWH.trans‐bent alignment have been characterized only theoretically to date and are still awaiting experimental confirmation.The V−V bond distances of 184.4 pm and 170.7 pm in V2H2, folded V2(μ‐H)2 and linear HVVH, are generated in solid Ne and identified by their IR spectra. UV irradiation induces the transformation of linear HVVH to folded V2(μ‐H)2. Irradiation with visible light leads to the transformation of the folded V2(μ‐H)2 isomer to linear HVVH. Quantum‐chemical calculations (MRCI) show that the folded V2(μ‐H)2 is the lower‐lying isomer with a 1A1 ground state. The linear HVVH with a 3Σg− electronic ground state is calculated to be at an energy of 0.48 eV with respect to the folded isomer. Both isomers are characterized by multiple V−V bonding interactions and the orbital occupations point to quadruple bonding. The calculated vibrational frequencies  support the assignment of the observed transitions. Although the folded isomer is the lower‐lying one, without irradiation, the linear isomer is formed in a substantial amount. This observation is consistent with the electronic ground states of the isomers. Since the V2 molecule has a 3Σg− ground state, the formation of the folded V2(μ‐H)2 isomer (1A1) by reaction of H2 and V2 is a spin‐forbidden process, whereas the formation of linear HVVH is spin‐allowed. Thus, it is reasonable that a substantial amount is trapped as HVVH. In ongoing work, we extend our work on other multiple‐bonded HMMH compounds. The experimental characterization of these small molecules is important for the detailed understanding of the bond properties and, thereby, also the reactivity of these systems.Multiple bonds between two transition metals are intensely studied due to their intriguing electronic structure and potential applications. The bond order is particularly high if the number of ligands is decreased to an absolute minimum. However, the synthesis of RMMR compounds  by conventional techniques in solution is a difficult task and, by all means, requires bulky ligands R. In this work, we demonstrate the use of the matrix‐isolation technique to experimentally characterize simple HMMH compounds with multiple MM bonds. Hence, two isomers of unprecedented VThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.