Alphaviruses are single stranded, positive sense RNA viruses that are often transmitted through mosquito vectors. With the increasing spread of mosquito populations throughout the world, these arboviruses represent a significant global health concern. Viruses such as Sindbis Virus (SINV), Chikungunya Virus (CHIKV) and Equine Encephalitis Viruses (EEV) are all alphaviruses. As viruses, these pathogens are dependent on the host cell environment for successful viral replication. It has been observed that viruses manipulate cellular metabolism and mitochondrial shape, activity, and dynamics to favor viral infection. This report looked to understand the metabolic changes present during Sindbis virus infection of hamster and human kidney cells. Cells were infected with increasing levels of SINV and at 24 hours post infection the mitochondria morphology was assessed with staining and mitochondrial activity was measured with a real-time Seahorse Bioanalyzer. The relative amount of mitochondrial staining intensity decreased with Sindbis virus infected cells. Both oxygen consumption rate and ATP production were decreased during SINV infection while non-mitochondrial respiration and extracellular acidification rate increased during infection. Collectively, the data indicates that SINV primarily utilizes non-mitochondrial metabolism to support viral infection within the first 24 hours. This understanding of viral preference for host cell metabolism may provide critical targets for antiviral therapies and help further define the nature of alphavirus infection. Alphaviruses are single stranded, positive sense RNA viruses. The genus alphavirus includes Sindbis Virus (SINV), Chikungunya Virus (CHIKV), Equine Encephalitis Viruses (EEV), and a variety of mostly-arthropod transmitted viruses . These vThere are limited options for treating alphaviruses. There is some published data showing that metabolic inhibitors such as imatinib mesylate can be used to reduce viral replication . By furtSindbis virus is often used as the prototype alphavirus . This wo2 in 25 cm3 cell culture flasks and split at 85% confluency (every 2-3 days) to maintain metabolic rates analyzed in experiments.Baby Hamster Kidney (BHK) (CCL-10) and Human Embryonic Kidney (HEK) (CRL-1573) were obtained from ATCC. Human hepatoma liver cells were obtained from Rushika Perera, who got them from the JCRB Cell Bank (JCRB0403 Huh-7). All cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% of a mix of Penicillin , Streptomycin , and Amphotericin (25 µg/mL) and 1% Ciprofloxacin (10 µg/mL). The cells were incubated at 37°C and 5% COCells were passaged, counted, and plated into 12, 24, or 48 well plates for the experiment. The default concentration of cells was 75,000 cells/mL. After 24 hours of the cells being attached and growing, a sample set of wells were counted using trypsinization and trypan blue quantification with a hemocytometer to determine the number of cells in each well. Virus was then added to the corresponding wells at the calculated multiplicity of infection . Virus stocks of Sindbis virus (AR339) and dsSINV were maintained at -80C and the stock was diluted using cell culture media. The diluted virus media at the specified concentration was then added to the cells at time 0 of infection. The infection proceeded for the allotted time until the treatment or experimental analysis was performed. Plaque assays were routinely performed to confirm the presence of infectious virus in viral stocks and samples. The reporter virus has been shown to accurately reflect infectious plaque forming units . 24 hourvia flow cytometry. BHK and HEK cells were cultured and allowed to grow for 24 hours. Those cells were then infected with Sindbis virus at various multiplicities of infection (MOIs) for 24 additional hours, and then incubated in a solution of 100uM Mitotracker for approximately 30 minutes before being run through the flow cytometer.To determine if there were any effects on the host cell mitochondria during Sindbis Virus (SINV) infection, the average density of Mitotracker fluorescence was measured Cells were plated in 24-well plates at 75,000 cells per mL and allowed to grow for 24 hours. The old media was then removed, and the cells were incubated in a solution of PBS and 100 µM green mitotracker dye  for 30 minutes. The cells were rinsed with PBS, trypsinized, and diluted with media, then transferred to 1.5 mL microfuge tubes. The cells were spun down at 2,000 xG for one minute at room temperature, the media was replaced with PBS, then run through the flow cytometer and the average green fluorescence per cell was analyzed. The Millipore Guavasoft flow cytometer was used for all experiments.Kidney cells were plated at 75,000 cells per mL, in a 48 well plate with 250 µL of cell suspension in each well. Serial dilutions of MFP  were made to test its efficiency as an antiviral treatment. The highest concentration used was 100 µM, and each concentration following was half of its previous. Cells were infected with dsSINV expressing mCherry  at vario4 cells per well in a 24 well plate  as in previous experiments and incubated as usual for 24 hours. Cells were infected with a MOI of 1 and incubated for one hour, followed by a change in media. An MOI of 1 was used to induce significant infection level (see Lactate accumulation over a 24 hour-period was measured in uninfected cells (MOCK) and Sindbis infected cells (SINV) using an enzymatic lactic acid assay kit . Solutions were prepared per protocol requirements and used for the following experimentation. Huh7 cells were passaged and plated at 6X102 infused incubator and cells were further processed through standard protocol and incubated for 24 hours before infection.Huh7 cells were passaged and plated as previously described with fresh 1X DMEM into a XFe24 plate . Huh7 cells were incubated for one hour in a humidified 5% CO2 incubator. Also, 48.8 mL of non-supplemented Seahorse XF base medium  was placed into a 37°C non-CO2 incubator for 1 hour.The day prior to the assay, after the 24-hour cell incubation period, cells were treated as described above. Further perpetration for the real-time metabolic analysis was needed and was done by placing 1 mL of calibrant solution  into the Seahorse sensor cartridge with drug ports  and was placed into a 37°C non-COOn the day of the assay, the 48.8 mL of Seahorse XF base medium was supplemented with 1 mM pyruvate by adding in 500 μL of 100 mM pyruvate , 2 mM L-glutamine by adding in 500 μL of 200 mM L-glutamine , and 10 mM glucose by adding in 200 μL of 2.5 M glucose  and pH to 7.4 with 0.1 M NaOH as per protocol for the Agilent mitostress test assay. This supplemented media was referred to as MST media.2 incubator for 1 hour. During the one-hour incubation period, the mitostress test drug compounds were prepared using the MST media per manufacturer protocol. The drug compounds at their given dry weight were 63 nmol of oligomycin, 72 nmol of FCCP, and 27 nmol of both rotenone/antimycin a. The drug compounds were combined with MST to make the following stock concentrations: 100 μM for oligo, FCCP and 50 μM for R/A. Oligomycin is an inhibitor of complex V of the electron transport chain and inhibits the synthesis of ATP, which decreases the oxygen consumption rate (OCR). FCCP uncouples oxygen consumption from ATP synthesis and increases OCR. Rotenone and antimycin-a inhibit complex I and III, respectively. R/A together inhibit the mitochondrion completely shutting down the ETC, in turn decreasing OCR. The working solution for these compounds were then made by adding additional MST media. The working solution for each MST drug compound were loaded into their respective drug ports. The port concentrations start at 10 μM for both oligo and FCCP, and 5 μM for R/A. The final well concentration after injections would be 1.0 μM for both oligo and FCCP, and 0.5 μM for R/A.Treated cells were processed by removing old media and washed two times with prepared MST media as per protocol recommendations. Cells then incubated in a non-CO2 incubator for one-hour, were analyzed on the Seahorse using the pre-set MST software protocol. After the metabolic analysis, cells were immediately trypsinized and a cell count was done for each well to determine the OCR measurements per total cells within each well. The OCR readings were normalized to the number of total cells within each well, using the control wells as the reference for each experiment.Before analysis of the cells, the cartridge plate with calibrant solution was placed into the Seahorse. After the calibrant stage and the cells incubated in a non-COOne way ANOVAs, T-test, and Tukey post-hoc comparison tests were performed to determine significance in data and results. All statistical analysis was done using the software R. Values shown in figures are the mean and the error bars represent the Standard error of the mean, unless otherwise noted. P < 0.001 is indicated by ***. P<0.005 is indicated by *.via flow cytometry to assess relative mitochondria mass or abundance. There was a decrease in the fluorescence given off by the mitochondria with higher levels of infection in both HEK and BHK cell lines (To determine if there were any effects on the host cell mitochondria during Sindbis Virus (SINV) infection, the average density of a mitochondrial fluorescence dye was measured ll lines Figure 1The Seahorse Bioanalyzer was used to determine respiration levels of the HEK cells during infection of various MOIs. This powerful instrument measures many variables that allow us to interpret where the energy within the cells is coming from and rule out pathways that the virus may or may not be using. Results showed a decrease in oxygen consumption rate (OCR) and an increase in extracellular acidification rate (ECAR) Figure 2After determining that oxygen consumption are affected during viral infection, it was important to determine what other mitochondrial characteristics were altered The OCR and ECAR values obtained from HEK cells using the Seahorse Bioanalyzer were used in calculating the basal respiration, maximal respiration, non-mitochondrial respiration, proton leak, ATP production, and spare respiratory capacity Figure 3SINV infection of HEK cells increases Non-mitochondrial respiration , compared to infected cells , Synthesis of ATP is dependent on Hvia fusion and is referred to as the Mitochondrial Fusion Promoter (MFP) It has been shown to act selectively on cells with fragmented mitochondria, and has no effect on wild type or healthy cells  antibiotic treatment. Interestingly, SINV-infected cells showed only a subtle drop in OCR, indicating a low level of oxygen utilization for ATP production. Treatment of mitochondria with MFP increased oxygen consumption, but had no effect on increasing virus replication, complementing our hypothesis that virus replication is not directly correlated with mitochondrial ATP production.With the increase in lactate accumulation and the reduction in mitochondrial metabolism, we can conclude that Sindbis Virus infection preferentially uses non-mitochondrial metabolism at 24 hour post infection of cultured cells.The original contributions presented in the study are included in the article/JC, JR, and JS conceived, designed, and orchestrated the work. AG and JS acquired funding and provided critical direction and oversight for the project. JC, JR, KY, and MS collected and analyzed the results. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.CBASE, Biology Department USAFA, Defense Threat Reduction Agency Service Academy Research Initiative (SARI).The views expressed in this article, book, or presentation are those of the author and do not necessarily reflect the official policy or position of the United States Air Force Academy, the Air Force, the Department of Defense, or the U.S. Government.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Extensive animal production systems are generally considered more sustainable and beneficial for the environment and for maintaining rural populations. However, there is no defined concept of what extensive milk production is. It is assumed to be a kind of production based on pasture and forage, with animals spending part of their daytime free and with a low stocking density. In order to increase consumer confidence in this type of product, we have studied markers based on molecules naturally present in the milk that allow us to differentiate the production system in which the cows that have produced the milk have come from. In addition, we are attempting to determine whether the milk production system can have benefits for consumer health.bta-mir-215, were higher in intensive systems than in extensive systems. Bovine mRNA targets for bta-miR-215 and their pathway analysis were performed. While the causes of these miRNAs differences remain to be elucidated, our results suggest that the type of production system could affect miRNAs levels and potential functionality of agri-food products of animal origin.Studying microRNA (miRNAs) in certain agri-food products is attractive because (1) they have potential as biomarkers that may allow traceability and authentication of such products; and (2) they may reveal insights into the products’ functional potential. The present study evaluated differences in miRNAs levels in fat and cellular fractions of tank milk collected from commercial farms which employ extensive or intensive dairy production systems. We first sequenced miRNAs in three milk samples from each production system, and then validated miRNAs whose levels in the cellular and fat fraction differed significantly between the two production systems. To accomplish this, we used quantitative PCR with both fractions of tank milk samples from another 20 commercial farms. Differences in miRNAs were identified in fat fractions: overall levels of miRNAs, and, specifically, the levels of  Consumers’ growing concern about food characteristics has contributed to the creation of a new concept of quality, particularly for animal products. This concept includes traditional attributes related to nutritional value, flavor, aroma, and color, together with new indicators related to ethical aspects, such as animal welfare and environmental impact of the production system . ConsumeMilk is a complex secretory product and a source of nutrients for children and adults. Milk also contains numerous biomolecules, including microRNAs (miRNAs), a group of non-coding RNAs that bind to specific regions within messenger RNA to regulate gene expression post-transcriptionally . In vitrChanges in diet have been shown to affect the expression of genes in the mammary gland ,17. ConsIn order to maximize potential differences in milk composition between intensive and extensive production systems, for sequencing, we sampled six farms at the end of one spring season (May 2016). On three farms (extensive production), animals grazed for at least for 12 h each day and ate a diet based on fresh grass with a small amount of concentrates. On three farms (intensive production), animals did not graze, instead eating a diet based on conserved feed and concentrates. Farm characteristics and details of the diets are described in For validation by RT-qPCR, twenty farms were sampled during autumn 2017 and spring 2018. Ten dairy farms applied an extensive system in which animals grazed and consumed a diet based on fresh grass, without maize silage and with a low amount of concentrates , while the other ten applied an intensive system in which animals were housed and ate a diet consisting of a high dry matter intake of maize silage  and a high amount of concentrate  .g for 20 min. The fat in the upper phase was transferred to a new 50-mL RNase-free tube. Then, 7.5 mL of QIAzol lysis reagent  was added, and the emulsion was vigorously mixed until the fat was well dispersed. The pellet (cellular fraction) from the initial centrifugation was washed twice with phosphate-buffered saline, then homogenized with 1 mL of QIAzol lysis reagent. All samples were stored at −80 °C until RNA extraction.From each type of farm, bulk tank milk was sampled after an even number of milkings (to avoid differences due to afternoon and morning milk composition). Samples were maintained at 4 °C and immediately transported to the laboratory for processing. Milk was well mixed, and 50 mL of each sample was centrifuged at 1900× For each sample, total RNA was extracted from 2 mL of milk fat in QIAzol lysis reagent, and from 1 mL of cell pellet resuspended in QIAzol lysis reagent. RNA was extracted using the miRVana miRNA isolation kit  following the manufacturer’s instructions, then stored at −80 °C. The concentration and integrity of RNA (RIN: RNA Integrity Number) for sequencing was further determined on an Agilent 2100 Bioanalyzer using an RNA 6000 Pico kit .6.In order to identify miRNAs differing between the two production systems, 12 libraries corresponding to the fat or cellular fractions of milk from six samples of bulk tank milk (three per production system) were prepared and sequenced using the Illumina platform  as 50 bp single reads. The raw sequence data were processed for quality control, and low quality reads were removed from raw data using CASAVA 1.8 based on chastity. Then, adaptors were trimmed, and sequences with read lengths between 15 to 40 nt were mapped to the bovine genome (bostau 7) and the miRNA database  in order to identify the known miRNAs. Expression levels of each miRNA were estimated based on the frequency of reads, and results were normalized to the number of reads per million (RPM) using the following formula: RPM =  × 10In order to select the miRNAs to be used as candidates for normalizers in RT-qPCR, we chose those miRNAs with more stable expression among samples for each milk fraction, that is, miRNAs with the smallest coefficient of variation (CV = standard deviation/mean).In order to identify those miRNAs whose levels differ between production systems, the results from miRNA sequencing were analyzed using three statistical tests. One test was the ratio of the difference between the means to the sum of the standard deviations between the two production systems: value = |(mean1–mean2)|/(standard deviation1 + standard deviation2) (strict Cohen’s d). This ratio indicates how many sums of deviations fit between the means; the higher the ratio is, the greater the difference between the means . AnotherA subset of the miRNAs which were determined to differ significantly between the two production systems identified in Total RNA was used for cDNA synthesis using the TaqMan Advanced miRNA cDNA Synthesis Kit , and the resulting cDNA was stored at −20 ℃ until use. Levels of miRNAs were determined using quantitative RT-PCR  in a StepOne thermocycler . The final reaction solution contained 10 μL of 2 × TaqMan Fast Advanced Master mix , 1 μL of 20× TaqMan Advanced miRNA Assay , 4 μL of RNase free water, and 5 μL of cDNA (diluted 1:10). The thermocycling program was set at 95 °C for 20 s, followed by 40 cycles at 95 °C for 1 s and 60 °C for 20 s. All PCR reactions were performed in duplicate, and a maximum of 0.5 threshold cycles were permitted between duplicates.Normalization is an essential component of a reliable qPCR assay. geNorm  is one op < 0.05.Levels of miRNA were normalized based on the geometric mean of the selected reference miRNAs selected by geNorm, estimated using QBase+ 3.1 software, Biogazelle   and exprhttp://www.pantherdb.org/ accessed on 8 June 2021) based on Gene Ontology classification [The Target Scan 7.2 bioinformatics tool  was usedfication .The total mean RNA concentration was 192.4 ± 37.4 ng/µL in milk fat and 30.6 ± 15.8 ng/µL (mean ± SD) in milk cells. The RIN value was 2.6 ± 0.15 for RNA from milk fat, and 6.5 ± 0.3 for RNA from milk cells.p = 0.004, p = 0.040, The six libraries from the cellular fraction of milk yielded a mean of 24,017,290.17 reads, significantly more than the 6,964,122.33 reads from the six libraries from the fat fraction (We identified 518 known miRNAs in the cellular fraction of milk and 477 in the fat fraction. Most of these miRNAs (454) were present in both fractions .The first 10 miRNAs in the fat fraction whose levels differed between the two production systems are ranked in Six miRNAs in each milk fraction were chosen as candidates for normalization . Followip = 0.030). The miRNAs bta-miR-2284y and bta-miR-2285e in the cellular fraction showed a trend towards lower levels in milk from extensive production, but the differences did not reach statistical significance.Based on analysis of the normalized miRNAs levels , the onlUsing Target Scan, 143 potential target genes were identified for bta-miR-215, which was abundant in intensive compared to extensive dairy production . Among tThe pathway analysis of bta-miR-215 target genes allowed for the identification of 41 associated biological pathways. The gonadotropin-releasing hormone receptor pathway and the Transforming Growth Factor β (TGF-β) signaling pathway  were higThe objective of this study was to identify a set of miRNAs in milk whose levels differed with the production system, in order to (1) determine whether miRNAs profiling can be used to authenticate milk from a given production system, and (2) investigate whether the production system can influence the functional properties of agri-food products such as bovine milk. To address these questions, we sampled milk from dairy farms in Asturias in northern Spain that applied intensive or extensive production practices, which differ significantly in numerous characteristics that can alter milk quality, including feeding management, animal density, and access to exercise through grazing .The origin of miRNAs in milk is controversial, and most miRNAs in milk are not found in blood . In addiWe successfully isolated total RNA from fat and cellular fractions of bovine milk, especially considering that the samples came from milk tanks on commercial dairy farms. The RIN was low for RNA from milk fat, which likely reflects its abundant content of low-molecular-weight RNA, which is different from ribosomal RNA .RNA sequencing analysis confirmed different RNA profiles in the fat and cellular fractions of bovine milk . InteresSequencing results did not allow for the identification of miRNAs that were specific to a given production system in either the fat or cellular fraction. Therefore, we focused on miRNAs whose levels differed between the two systems, and we validated a subset of the promising miRNAs using quantitative RT-PCR. The only miRNA that we validated to differ significantly between the two production systems was bta-miR-215 in the fat fraction . This miAmong the 143 genes identified by bioinformatic tools, Fabp3 stands out for its involvement in fatty acid transport and activation in the bovine mammary gland . Vargas-Apart from Fabp3, another two genes are of particular interest: Mapk1 and Bmpr2. Mapk1 is involved in the regulation of milk protein synthesis , and BmpWe also identified the miRNAs bta-mir-2284y and bta-mir-2285e in the cellular fraction of milk whose levels tended to differ between intensive and extensive production systems. Further analysis showed that these differences became significant when we compared animals on diets containing more or fewer than 10 kg of concentrates per day (data not shown). These results suggest that our failure to detect significant differences in levels of bta-mir-2284y and bta-mi-2285e in the overall analysis may reflect milk sampling from commercial farms, as well as the difficulty of defining intensive and extensive production. Further studies should examine these two miRNAs as additional potential biomarkers for authentication and functional analysis of agri-foods.Finally, our study presents several limitations. The study was carried out in commercial farms, where managing conditions and diets are quite different even within the same group. Validation in controlled conditions at experimental farms, which reduce internal variance of the experimental groups, should help to identify miRNAs as putative biomarkers for dairy production systems. In vitro experiments will help to elucidate the functional features of milk produced under different dairy systems.We investigated differences in miRNA profiles of raw cow tank milk from commercial farms which applied either intensive or extensive production systems. We identified bta-miRNA-215 in the fat fraction of milk as a possible biomarker of milk from intensive production systems. Our results imply that the type of production system can influence miRNA levels, and, therefore, functional properties of bovine milk, as well as, potentially, other agri-food products of animal origin.


Adenocarcinoma of the urinary bladder is an extremely rare entity. It has aggressive behavior and it responds poorly to chemotherapy and radiotherapy. Painless hematuria and passage of clots are mainly common symptoms. Primary bladder adenocarcinoma has a poor prognosis due to its late presentation at an advanced stage. We present here, a rare case report of a 65-year-old male who presented with a huge ulcerative fungating tumor of clinical size 7 x 6 x 5 cm and radiological size 8.6 x 8.1 x 8 cm (exophytic component) and 3.6 x 3.3 x 3 cm (endophytic component) at suprapubic region eroding through pubic bone, rectus muscle and skin with features of lung and inguinal nodes metastasis. Wedge biopsy from fungating growth was suggestive of adenocarcinoma of the urinary bladder of papillary mucin secreting subtype which is also a very rare variant and subtype to get reported. It has a very poor prognosis and only a 6% of survival rate. Keeping all this context, due to poor Eastern Cooperative Oncology Group (ECOG) performance status, huge tumor burden, and advanced presentation of disease, it was suggested by the institutional tumor board, to provide him with the best palliative care management. Cancer of the bladder, also known as urothelial cancer or urinary bladder cancer, is the 10th most common cancer in the world and its incidence is steadily rising worldwide, especially in developed nations . In the A 65-year-old male presented to Urosurgery Outpatient Department (OPD) with complaints of a huge supra pubic lump for four months, which ulcerated into a fungating mass over a period of four weeks; ripping off, protruding, and eroding through all suprapubic structures. He also complained of hematuria for 1.5 months which was insidious in onset and rapidly progressive in nature. He also had associated symptoms of increased frequency of micturition and burning micturition for 10 days. He had a history of nausea and vomiting, no history of fever, pain in the abdomen, breathlessness, jaundice, backache, or any bony ache. He also has no history of any comorbidity . He had a past history of open cystolithotomy done eight years back.On clinical examination, the patient was poorly built with an Eastern Cooperative Oncology Group (ECOG) performance status of 3. He also had significant pallor present but no icterus, cyanosis, or clubbing. Clinically, the patient had a right inguinal lymph node palpable. He was vitally stable. On per abdominal examination, there was a single ulcerative fungating mass of size 7 x 6 x 5 centimeter (cm) in the suprapubic region, fixed to the underlying muscle and bone. It was firm in consistency, friable surface, bleed on touch with right inguinal lymphadenopathy of size 2.5 x 1.5 cm. The mass was non-tender, firm in consistency, and mobile in the vertical direction , creatinine 2.6 mg/dL, sodium 141 milli equivalent per liter (mEq/L), and potassium 4.8 mEq/L. Since his creatinine was raised; patient underwent non contrast computed tomography of abdomen which was suggestive of large exophytic polypoidal lobulated soft tissue mass lesion noted in the pelvis with loss of fat planes with antero-superior wall of the bladder, arising from the urinary bladder. The lesion is protruding into the lumen of urinary bladder, suggestive of endophytic component. Bulk of the lesion is present in anterior perivesical space with extension outside the anterior abdominal wall. Posteriorly, the lesion is involving antero-superior wall of urinary bladder and bilateral vesico-ureteric junction with resultant bilateral hydroureteronephrosis. Laterally, the lesion is abutting proximal portion of bilateral spermatic cord. There is mild perivesical and perilesional fat stranding. There is a maintained fat plane between the lesion and seminal vesicles. The exophytic component of lesion measures 8.6 x 8.1 x 8 cm. Endophytic component of the lesion measures 3.6 x 3.3 x 3 cm  was done and palliative treatment was advised, considering ECOG performance status of patient to be 3, deranged kidney function and huge tumor burden with advanced presentation of the adenocarcinoma of bladder with multiple right inguinal lymphadenopathy and distant lung metastasis. Adenocarcinoma is a rare histological variant that accounts for 0.5%-2% of urinary bladder carcinoma in the United States . PatientThe pathogenesis of bladder adenocarcinoma in although not yet understood but mostly, almost 90% of bladder tumor patients with exstrophy growth of the bladder are adenocarcinoma . AccordiThe association between urinary bladder stones with SCC of the bladder is well established in the literature and vesical stones have been considered a risk factor for SCC of the bladder -22. AlshOn histological examination, adenocarcinoma of the bladder may show various growth patterns like (i) enteric  (5%); (ii) mucinous/colloid 15%); third most common (iii) signet ring cell (22.5%); second most common; (iv) not otherwise specified (NOS) (52.5%); most common; and (v) mixed pattern ,15. In o%; third Shinagare et al.  have demMetastasis to inguinal lymph nodes in cases of bladder cancer is very rare scenario. Only few cases (4-5) of such inguinal metastasis have been reported in the literature and that to after radical cystectomy in cases of transitional or urothelial carcinoma of bladder ,27. PatiMost of the primary bladder adenocarcinoma patients have muscle invasion and are treated with radical cystectomy and pelvic lymph node dissection ,29. SeveThe prognosis and outcome of adenocarcinoma of bladder is very poor due to late diagnosis of its advanced stage . The surAdenocarcinoma of the urinary bladder is an aggressive type of bladder tumor. In the literature, only a few cases exhibiting association of adenocarcinoma bladder with bladder stones have been reported. Our patient’s unusual presentation with a history of bladder stone removal in past and adenocarcinoma of the bladder highlights the possible interrelation between these two conditions.Presentation of bladder tumors (of any type) in the form of such a huge fungating mass eroding, splitting up, and protruding through suprapubic structures has never been reported yet. This might be the first such case to get reported with such a rare clinical presentation. While dealing with the cases of bladder cancers, awareness regarding this variant, adenocarcinoma with a mucinous subtype of bladder cancer must be kept into consideration.The treatment guidelines or protocol for managing adenocarcinoma of the urinary bladder varies according to the muscle invasion and staging of the tumor. To date, limited available data suggests adenocarcinoma is an aggressive type of bladder tumor with poor clinical outcomes and prognosis.


To facilitate the use of in silico data in regulatory submissions, explanatory control compounds should be tested and documented to demonstrate consistency between predictions and the historic validation data. This study aims to quantify drug-induced electrophysiological effects on in silico cardiac human Purkinje cells, to compare them with existing in vitro rabbit data, and to assess their accuracy for clinical pro-arrhythmic risk predictions. The effects of 14 reference compounds were quantified in simulations with a population of in silico human cardiac Purkinje models. For each drug dose, five electrophysiological biomarkers were quantified at three pacing frequencies, and results compared with available in vitro experiments and clinical proarrhythmia reports. Three key results were obtained: 1) In silico, repolarization abnormalities in human Purkinje simulations predicted drug-induced arrhythmia for all risky compounds, showing higher predicted accuracy than rabbit experiments; 2) Drug-induced electrophysiological changes observed in human-based simulations showed a high degree of consistency with in vitro rabbit recordings at all pacing frequencies, and depolarization velocity and action potential duration were the most consistent biomarkers; 3) discrepancies observed for dofetilide, sotalol and terfenadine are mainly caused by species differences between humans and rabbit. Taken together, this study demonstrates higher accuracy of in silico methods compared to in vitro animal models for pro-arrhythmic risk prediction, as well as a high degree of consistency with in vitro experiments commonly used in safety pharmacology, supporting the potential for industrial and regulatory adoption of in silico trials for proarrhythmia prediction.The preclinical identification of drug-induced cardiotoxicity and its translation into human risk are still major challenges in pharmaceutical drug discovery. The ICH S7B Guideline and Q&A on Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential promotes human  Kr), which - when blocked - leads to prolongation of the QT segment of the ECG, and potentially to arrhythmia. Predicting proarrhythmia is challenging, due to the interplay of different ionic currents underlying the cellular electrical activity, i.e., the action potential (AP), and the complex drug-ionic channels interactions.Preclinical assessment of drug-induced arrhythmia or proarrhythmia is a key requirement for pharmaceutical industries and regulators. This is particularly relevant for compounds showing a positive hERG (human Ether-à-go-go-Related Gene) signal, but also blocking other ionic channels . The curIn vitro, in vivo, and ex-vivo animal models are widely used for preclinical proarrhythmia assessment, often considering metrics based on drug-induced AP prolongation as a surrogate of QT prolongation. Among these, cardiac Purkinje fibers obtained from dog or rabbit hearts, are one of the most established and ICH S7B-recommended in vitro models for preclinical cardiotoxicity screening  initiative  for four cardiac ion channels, for each compound , the L-type calcium channel hCav1.2 , and the sodium channel hNav1.5 . We also considered the potassium channel hKv4.3  as IC50s were also available for this channel.The CiPA Ion Channel Work Group has investigated six cardiac ion channels of potential relevance for human QT effects. From those, we employed for this study four key human cardiac ion channels, including the minimum set of ion channels required for reliable dictions . This miExperimental testing of ion channel inhibitions of hERG, hCav1.2, hNav1.5 and hKv4.3 was performed on engineered immortalized cell lines using patch clamp protocols at either room (21°C) or physiological (33°C) temperature. However, since ion channel inhibition data recorded at physiological temperature often show much greater inherent variability due to complexity of the protocol, only data generated at room temperature were considered throughout this study. Also, for the reference set of compounds, the room temperature protocol was found to better agree with published data .in vitro hERG testing, hERG-CHO (Cytomyx CYL3038) were cultured in glutamax DMEM/F12 medium containing 10% fetal bovine serum and 1% G418 in a humidified, 5% CO2 atmosphere. Cells were grown at 37°C, then transferred to a 30°C incubator at least 24 h before the experiments to improve the hERG expression. HEK-293 cells were transfected stably with hERG cDNA and cultured in a 50:50 mix of Dulbecco’s Modified Eagle’s Medium and Ham’s Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS). A robotized planar patch clamp platform  was used, reproducing the experimental quality standards and analysis routines prevailing in manual patch clamp . The patch clamp protocol involved a −80 mV holding potential, then cells were depolarized to 20 mV for 5 s, followed by a 5 s deactivation pulse to −50 mV to reveal hERG tail current. This paradigm was delivered once every 15 s to monitor current amplitudes. Each concentration was tested in a standard protocol for at least 3 cells (n ≥ 3). Starting from −80 mV, effects of sample compounds on the onset and steady state inhibition of hERG current were followed in response to a repeated pulse pattern, damping from steady +20 mV to −80 mV. Obtained raw data was corrected for external effects at saturation concentration. Each compound was tested at least in triplicates at six appropriate concentrations in ascending order, and concentration limits were defined based on compound solubility, cytotoxicity, or compound physicochemical properties.For in silico trials are reported in For the other ion channel targets  the screening was performed on the QPatch platform . HEK-293 and CHO (for Cav1.2) cell lines expressing exogenous human targets were cultured according to internal protocols, in DMEM/F12 media supplemented with 10% FBS. Standard 48 well plates were used in all experiments, and a standard voltage protocol mimicking elements of a ventricular action potential was applied at eight increasing concentrations to facilitate the determination IC50 values, with replicates. If no half-maximal inhibitory concentration was achieved in the specified concentration range, the result was interpreted as ‘no inhibition’. Missing or inconclusive data in our studies were complemented from literature . All IC5in vivo ECG. The effects of the 14 reference compounds on cardiac Purkinje electrophysiology were previously evaluated through microelectrode recordings from isolated rabbit Purkinje fibers . Details of the preparation and conduction of the studies have been previously described  to obtain a stock solution. This solution was further diluted into 100% DMSO to obtain solutions at different concentrations , AP duration at 50% and 90% of repolarization (APD50 and APD90 in ms).The following biomarkers were quantified: take-off potential , i.e., the membrane voltage preceding phase 0, AP amplitude , maximal upstroke velocity . After testing the highest concentration, the physiological solution was superfused again to evaluate the reversibility of the drug effect.in silico model (www.cs.ox.ac.uk/insilicocardiotox/model-repository). As shown in + currents , ICaL, T-type Ca2+ current (ICaT), Ito, sustained outward K+ current (Isus), rapid and slow delayed K+ rectifiers , inward K+ rectifiers (IK1), funny current (If), Na+-Ca2+ exchanger (INCX) and Na+-K+ pump (INaK). The model was calibrated and evaluated against a wide set of AP recordings from human healthy Purkinje cells in control conditions and after exposure to dofetilide, and it is also able to reproduce the most common arrhythmia precursors at the cellular level, i.e., early and delayed afterdepolarization .Human cardiac Purkinje electrophysiology was simulated using the Trovato2020 co model , publicl© 2018 Oxford University Innovation Ltd. Oxford, UK), a user-friendly software to perform in silico drug trials in population of models  in the range [50–200]% of their baseline values, using Latin hypercube sampling , and additionally the AP duration at 10%, 25%, and 75% of repolarization  and the “end of potential” voltage (EOP). Only models exhibiting all AP biomarkers within the experimental ranges measured in healthy human Purkinje cells  Core (TM) i5-4670S CPU @ 3.10 GHz RAM: 8 GB, 64-bit Windows 10). The time required to simulate one drug at one concentration (150 beats) in a population of 530 models was 12, 17, and 35 min, for simulations at 3, 1, and 0.2 Hz, respectively.in silico and in vitro models. Then, we benchmarked the results for each compound at each concentration and pacing frequency against control. Results are shown as percent variations of the means, for both experiments and simulations.We first compared the mean of simulated and experimental AP biomarkers in control conditions (no drug) for all pacing frequencies, to assess the consistency between the in silico population or 1 cell of the in vitro rabbit assay, at any of the tested concentrations, were classified as risky, whereas they were classified as safe if all models/cells fully repolarized at slow pacing. However, differently from  qualitative agreement, if the trend (increase/decrease) was the same, but the difference between the in vitro and in silico means higher than 15%; 3) disagreement, if the trend was different, regardless of the magnitude of the mean difference.Finally, to evaluate the consistency between experiments and simulations, we applied a third metric, based on the mean of drug-induced percent variations in AP biomarkers for each tested drug and concentration: 1) strong agreement, if the trend (increase/decrease) was the same, and the difference between the in vitro rabbit experiments (n = 74) and the population of human in silico models (n = 530) at slow, normal, and fast pacing. APD90 (and to a lesser extent APD50) is larger in rabbit experiments compared to human simulations at all pacing frequencies and, to a greater extent, at slow pacing. Decreasing the pacing frequency results in larger APD prolongation in rabbit than human Purkinje cells, in agreement with what has been previously measured in rabbit , while the in vitro rabbit models achieved an accuracy of 79%. Indeed, in rabbit preparations, EADs were observed for only 8 out of 11 risky compounds, while no EADs were observed for bepridil, ranolazine, and terfenadine, despite the wide range of concentrations explored. Diltiazem, nifedipine and verapamil did not induce any EADs in silico nor in vitro and were correctly classified as safe.The occurrence of drug-induced abnormalities in repolarization was quantified in both experiments and simulations as reported in CaL  and were therefore classified as risky. This AP prolongation is due to the large (>50%) hERG blockade at concentrations far from the EFTPCmax. In vitro rabbit assays produced the same overall accuracy, even though it was achieved with one false positive (verapamil) and one false negative (terfenadine), the latter showing little AP prolongation at all tested concentrations tested (up to 593x EFTPCMAX), despite its high TdP risk.Since APDin silico and in vitro predictions based on APD90 prolongation at 1 Hz: out of 14 reference compounds, 11 were in agreement, 2 were in partial agreement, and only 1 was in disagreement . A similar behavior was also observed for sotalol , limited by the solubility of the drugs. This limitation can be easily overcome with in silico trials, and–when simulating higher concentrations–we observed larger AP prolongations in the population of models: +57% for dofetilide 0.1 µM and +39% for sotalol at 100 µM.Partial agreement was observed for clarithromycin and dofetilide. As shown in  sotalol , and it  sotalol , i.e. laMAX show an almost total agreement : the only drug showing disagreement was nifedipine at 10 μM, which slightly increased dV/dtMAX (4%) in rabbit and reduced it in humans . Drug-induced APD90 changes also showed a high degree of consistency (94–96%) between experiments and simulations, even though - compared to dV/dtMAX - a larger portion of drugs showed qualitative rather than strong agreement. Disagreement was only found in 6%, 5%, and 4% of drug concentrations, for slow, normal, and fast pacing, respectively. Results for APD50 also showed good agreement (73–83%), despite the percentage of disagreement being higher compared to dV/dtMAX and APD90. This difference could be explained by the differences in AP morphology between humans and rabbit: rabbit APs have a more pronounced spike compared to human in silico models and this affected the voltage threshold to compute the APD50 , using in silico human Purkinje models with a variety of ionic profiles (n = 530), their comparison to preexisting in vitro rabbit Purkinje fibers experiments (4 ≤ n ≤ 6 for test compound), and their ability to predict clinical risk, based on various biomarkers, including EADs occurrence and APD90.In this study, we showcase the large impact that human In silico predictions using human Purkinje models based on EAD occurrence at slow pacing showed 100% accuracy in classifying risky from safe drugs, while in vitro rabbit experiments yielded 79% accuracy. This was also superior to predictions based on AP prolongation, which yielded accuracy of 86% for both in silico and in vitro.1 In silico drug trials using human cardiac Purkinje electrophysiology models and in vitro rabbit Purkinje recordings showed a high degree of consistency for all tested compounds, across biomarkers, concentrations and pacing frequencies. This supports the credibility of human-based in silico modelling and simulations for the replacement of animal experiments in this context of use.2 in vitro rabbit compared to in silico human recordings. This agrees with well-known differences between rabbits and humans in the response to hERG block.3 Three compounds, i.e., clarithromycin, dofetilide, sotalol, displayed a larger AP prolongation The main findings of this study are:in silico drug trials to clinical outcome - as demonstrated here for electrophysiology–highlights their potential for high regulatory impact in drug discovery  for animal research  rabbit Purkinje fibers are commonly used as an in vitro model to predict proarrhythmic risk in human. Therefore, here we evaluate how the human-based in silico methodology performs compared to commonly used experimental models. This is particularly relevant for the 3Rs, as it allows reusing data. 2) the ultimate goal of preclinical studies is to predict proarrhythmic risk in humans, and human-based models that capture human patho-physiology are crucial to overcome some of the limitations of animal models. Using combinations of experimental and computational methods effectively require the evaluation of their consistency as performed here.Also, in this study we decided to compare human in silico drug trials, compared to only 86% using in vitro recordings, which failed to identify two compounds with known TdP risk (bepridil and terfenadine) and one with conditional TdP risk (ranolazine). Our findings agree with previous experimental studies, showing that these three compounds often did not show EADs when tested in rabbit preparations. Bepridil up to 10 µM did not induce EADs in rabbit hearts . In addition, the in silico population of models incorporates a large variability in ionic profiles (over- and under-expression of ionic currents), and we previously demonstrated that models with high repolarizing currents and low repolarization reserve are more likely to develop EADs following ion channel blocks, both in ventricular and cardiac Purkinje models  or very rare mutations (resulting for example in low conductance of a particular ionic current). This information is important for decision making and could guide further experiments. No EADs in the simulations are strong digital evidence supporting the safety of the compound, as far as direct effects of the compound to ion channel conductance are concerned. However, this result can be due to the lack of direct effect of the compound, and indirect effects must be ruled out by different experiments, as it is outside of the context of use defined for the in silico studies.in silico drug safety assessment, consider other tissue models as human stem cell-derived cardiomyocytes , algorimyocytes , and appmyocytes  and/or smyocytes .in silico and in vitro predictions based on drug-induced AP prolongation compare against clinical risk. Although several studies showed that APD increase is not always associated with TdP risk, especially for multichannel blockers (90 at 1 Hz reached the same accuracy (86%) in silico and in vitro, and results were highly consistent (>90%) across all drugs and all concentrations.We also evaluated how blockers , it is sMAX, APD90, and APD50) and pacing frequencies between human simulations and rabbit preparations also showed large consistency. This is a very exciting result, confirming the importance of developing in silico models using experimental data at different frequencies, as we did for the human cardiac Purkinje models used in this study  to demonstrate high grade of consistency between simulations and experiments. Our results showed not only high degree of consistency between in vitro and in silico preparations arations , but alsarations  since thin silico model compared to perform in vitro animal experiments. These include: 1) reduction of the use of animals in research; 2) reduction of the time required for drug safety assessment, thus allowing pharmaceutical companies to process more compounds in a shorter amount of time, and accelerate the drug development process; 3) economical advantage, i.e., in silico trials can be performed in a standard computer; 4) overcome limitations in the tested concentration ranges, due to drug solubility problems; 5) overcome limitations in the number of conditions explored for each drug  and in the number of samples.In addition, there are more advantages in using in silico drug trials in human cardiac Purkinje cells have shown to be consistent with in vitro recordings from rabbit Purkinje fibers, and more accurate for predictions of drug-induced proarrhythmic risk. This study contributes evidence towards the vision of in silico trials for preclinical drug assessment as alternative to animal models.To conclude,


Acipenser baerii) and hybrids of Siberian sturgeon and Russian sturgeon (Acipenser baerii × Acipenser gueldenstaedtii). The embryo survival rate was higher in group B hybrids i.e., ♀ [Siberian sturgeon × Russian sturgeon] × ♂ Siberian sturgeon than in the control group (Siberian sturgeon), whereas group A hybrids i.e., ♀ [Siberian sturgeon × Russian sturgeon] × ♂ Siberian sturgeon were characterized by lower survival rates than the group C embryos (control). These results suggest that the survival of hybrid embryos could be determined by individual traits, such as gamete (eggs and sperm) quality and genetic factors. Sturgeon (Acipenseridae) fish are a valuable fish in aquaculture not only because of the taste of their meat, but also because of the caviar obtained from the production process. Sturgeon species differ from teleosts in the main stages of the life cycle, in particular embryonic development. Therefore, research regarding the controlled reproduction and egg incubation are key during breeding and hybridization programs of sturgeon species. Moreover, sturgeon hybrids are characterized by high values of performance, breeding, and reproductive parameters, and their rearing is often more economically efficient than the rearing of purebred species Therefore in this study we analyzed selected stages of embryonic development in Siberian sturgeon (Acipenser baerii) and hybrids of Siberian sturgeons and Russian sturgeons (Acipenser baerii × Acipenser gueldenstaedtii). For this purpose, embryos representing nine distinct developmental stages  were sampled from each group  during incubation. Stages of embryogenesis were identified based on a 30-point scale of embryonic development in sturgeons. A total of 13 developmental stages were identified, including early cleavage, blastula formation, early and late gastrulation, onset of neurulation, beginning of organogenesis, and prelarvae. During gastrulation, the survival of hybrid embryos was highest in group B (93.8%) and lowest in group A (86.7%). Embryonic deformation was not observed during experimental incubation. The archived data relating to the embryonic development of Siberian sturgeon × Russian sturgeon hybrids could be applied to identify the individual stages of embryogenesis in hybrid sturgeons during egg incubation.The aim of this study was to describe the selected stages of embryonic development in Siberian sturgeon ( In the northern hemisphere, sturgeons (Acipenseridae) have been long regarded as valuable fishery resources, mainly in caviar production. In recent decades, anthropogenic and industrial activities posed a serious threat to the natural habitats of wild sturgeon populations . Most st1 hybrids produced by crossing species with a similar number of chromosomes proved to be particularly successful. The following F1 hybrids were obtained: beluga × sterlet, Siberian sturgeon × Russian sturgeon, as well as backcrossed hybrids, such as [Siberian sturgeon × Russian sturgeon] × Siberian sturgeon [The first ichthyological research to support natural sturgeon populations involved artificial breeding and broodstock production (mostly fry) for open-water stocking. In successive stages, the scope of the research was expanded to include fry and fingerling rearing under controlled conditions, the selection of breeding candidates, and the establishment of the sturgeon spawning stock. In vivo methods for collecting gametes were developed, which enabled multiple uses of the same spawners ,5, selecsturgeon . RussianAcipenser baerii Brandt, 1869 × Acipenser gueldensaedtii, Brandt and Ratzeburg, 1833) hybrid females with sperm collected from Siberian sturgeon males. Embryonic development stages were compared in backcrossed hybrids and purebred Siberian sturgeons.In Poland, various sturgeon species and hybrids are artificially bred to produce broodstock for further rearing. Therefore, a sound knowledge of embryonic development in purebred sturgeon and hybrids is needed to monitor all stages of the process and detect any abnormalities. The aim of this study was to analyze the embryonic development of backcrossed hybrids obtained by fertilizing eggs collected from Siberian sturgeon × Russian sturgeon (Embryos (hybrids or pure species) for the study were obtained from female A and female B—Siberian sturgeon × Russian sturgeon hybrids (hybrids), and female C—purebred Siberian sturgeon (control group). Eggs collected from all three females were fertilized with sperm collected from Siberian sturgeon males.−1), and males were treated with the Ovopel hormonal treatment at 1 granule per kg−1. Eggs were sampled in vivo, and sperm was collected by a syringe with an elastic cannula. Sperm quality was evaluated under a microscope to evaluate the sperm activated in water. Eggs were fertilized by the semidry method with sperm diluted in water at a 1:50 ratio. Fertilized eggs were incubated at 16 °C (±0.5 °C) in Weiss mini-incubation chambers in a recirculating aquaculture system (RAS) in the Wąsosze Fish Farm, near Konin. Dissolved oxygen, water temperature, and pH were maintained at 9.5 mg L−1, 16 °C, and 8.0, respectively, throughout the experimental period. The mean concentration of ammonium nitrogen and nitrites contents was below 0.01–0.05 mg L−1 throughout the experimental period. The photoperiod during the experimental trial was maintained at 12L:12D.Sturgeons were bred in the Wąsosze Fish Farm, near Konin. One Siberian sturgeon female, two Siberian sturgeon x Russian sturgeon hybrid females, and three Siberian sturgeon males were prepared for breeding. The fish were initially subjected to environmental stimulation and then stimulated with hormones upon the achievement of gonadal maturity stage IV . FemalesThe fertilized eggs from three females were incubated in three groups , and 20 embryos representing nine distinct developmental stages  were sampled during incubation. Embryos were sampled (nine sampling sessions) from groups A, B, and C . Every sample from each group  was placed in 4% paraformaldehyde in Eppendorf tubes labeled with a letter denoting the female  and the number of the sampling session (1–9). The samples were placed on Petri plates and observed under the Olympus SZX16 stereoscopic microscope  equipped with the Olympus DP25 camera . The Olympus KL2500 LCD  fiber optic light source was used for optimal sample imaging. The samples were observed, and images were acquired with the use of Olympus CellSens Dimension software .Acipenser gueldenstaedtii) compiled by Dettlaff and Vassetzky [The stages of embryonic development were identified by analyzing the structures characteristic of each stage. A list of 36 distinct stages of embryonic development in Russian sturgeon  was provided.The results of survival are presented as the mean ± SD, and the differences were considered significant at Images of each embryonic development stage were acquired for analysis . No diffSturgeon hybrids are characterized by high values of performance, breeding, and reproductive parameters, and their rearing is often more economically efficient than the rearing of purebred species ,11. The Controlled breeding and egg incubation are key stages during breeding and hybridization programs. During incubation, embryos proceed through a series of developmental stages, and larvae are hatched. Embryogenesis and successive developmental stages should be closely monitored to identify any irregularities. In purebred sturgeons and hybrids, the main stages of embryonic development are highly similar, but sturgeon species have different thermal requirements, which can affect the rate of embryogenesis. Therefore, embryonic development in purebred sturgeon and hybrids should be thoroughly investigated to monitor all stages of the process and identify any abnormalities. In comparison with teleosts, sturgeon species differ in the main stages of the life cycle, in particular embryonic development ,10. SturDevelopmental defects can be observed in different stages of embryogenesis. According to Dettlaff et al., , most deMost developmental defects result from abnormalities in the earliest stages of embryogenesis (oocyte maturation and fertilization). Research into several sturgeon species has demonstrated that abnormalities in later stages of embryogenesis can be caused by inadequate environmental conditions and poor water quality ,14,18. TAcipenser baerii) has been previously analyzed by Park et al., [Acipenser gueldenstaedtii), with some modifications. Dettlaff and Vassetzky [In the current study, Siberian sturgeon embryos and the embryos of Siberian sturgeon × Russian sturgeon hybrids developed normally. The embryonic development of Siberian sturgeon , whereas group A hybrids were characterized by lower survival rates than group C embryos. These results suggest that the survival of hybrid embryos could be determined by individual traits, such as gamete (eggs and sperm) quality and genetic factors. Eggs’ quality was assessed as very high based on morphological traits, and the influence of genetic factors could not be ruled out. In view of the above, further research is needed to examine the genetic profile of the parent species  selectedCurrently, the production of sturgeon fish shows an increasing trend. In addition to pure species, interspecies sturgeon hybrids are also bred. The production of sturgeon hybrids is important in aquaculture because these hybrids very often show favorable breeding traits that are desired by fish farmers. This paper describes the embryonic development of Siberian sturgeon × Russian sturgeon, which is the most common interspecies hybrid used in European aquaculture. During the trial, the most important developmental stages were described, and based on these descriptions it is possible to confirm the accuracy of the embryogenesis of the studied hybrids.


The domestic cat is the world's most popular pet and one of the most detrimental predators in terrestrial ecosystems. Effective protection of wildlife biodiversity demands detailed tracking of cat trophic ecology, and stable isotopes serve as a powerful proxy in dietary studies. However, a variable diet can make an isotopic pattern unreadable in opportunistic predators. To evaluate the usefulness of the isotopic method in cat ecology, we measured C and N isotope ratios in hundreds of archaeological cat bones. We determined trends in cat trophic paleoecology in northern Europe by exploiting population-scale patterns in animals from diverse locations. Our dataset shows a high variability of isotopic signals related to the socio-economic and/or geomorphological context. This points toward regularities in isotopic patterns across past cat populations. We provide a generalized guide to interpret the isotopic ecology of cats, emphasizing that regional isotopic baselines have a major impact on the isotopic signal. They can adjust to various conditions and successfully colonize a broad spectrum of environments3. Even when fed enough by their human owners, they search for other food sources and easily become feral (fully independent of people)3.From an ecological perspective, the domestic cat is a very flexible animal. Despite being a hypercarnivore , cats select prey opportunistically4. Its domestication and rodent-controlling ability, followed by its global dispersal, is essential for our understanding of the diversity and evolution of human–animal relationships and the history of husbandry10. The domestic cat has become a widespread and abundant predator, and is considered one of the most detrimental species in the world that negatively impacts a wide range of wild native species14. Domestic cats also pose a serious threat to wildcat native populations through hybridization16 and competition18. Thus, the study of domestic cat ecology is important from various points of view—from the history of one of the world's most popular pet to conservation biology.The history of the domestic cat and its ecological adaptations are currently highly debated topics20. The Near Eastern ancestor of domestic cats had already emerged in Central Europe 6000 years ago; however, the possible relationships of this early population with modern domestic cats are unknown10. Still, the most plausible hypothesis assumes that the Roman Empire, with its military campaigns and far-reaching trade, played a major role in establishing a permanent domestic cat population across Europe22. However, archaeological evidence indicates that cats only became a constant and frequent component of the European anthropic landscape during the Middle Ages22. Viking expansion, followed later by trade contacts with the Ottoman Empire, played an important role in further distributing cats across the continent, and establishing the modern-day population23. During this period, cats became important pest controllers, and their numbers increased in parallel to the expansion of towns22. The high concentration of mice and rats and easy access to human food waste allowed dense cat populations to develop24.The earliest history of the human–cat relationship is still poorly documented, due to the sporadic occurrence of cat bones in archaeological and paleontological contexts, with records from the early stages of human–cat relationships containing long time gaps27. For decades, this method has been used to detect past diets, providing insights into animal trophic ecology and adaptation to the anthropogenic landscape29.The possibility of using stable isotopes to reconstruct diet became a powerful tool in archaeological studies focused on elucidating past human and animal ways of life2. However, because it is a hypercarnivore, the bulk of potential food sources is relatively well defined. Until now, only few studies have investigated the ancient or modern cat diets using stable isotopes34. Here, we aimed to investigate the pattern of carbon and nitrogen isotopic variability in domestic cats from Northern Europe. Through analyzing a vast database of bulk collagen δ13C and δ15N values and comparing environmental and cultural conditions, we explore: (i) the range of isotopic variability among cats; (ii) predictability of cat isotopic signal in the context of its environment and presumed ecology; and (iii) usefulness of the stable isotope method to monitor the ecology and diet of this opportunistic and individualistic carnivore.The domestic cat is a complex object for stable isotope analysis because it opportunistically selects a wide range of food sources36. Our material originated from northern Europe, which is a region large enough to represent variable landscapes and socio-economic contexts, but is sufficiently restricted to avoid covering extremely different ecosystems, which could dominate the isotopic ecology and obscure minute differences.We used a collection of archaeological cat bones from medieval and post-medieval Europe, ranging from the 6th to the eighteenth centuries AD Datasets  and S2. Cat remains are scarce at archaeological sites. Complete or partial carcasses occur sometimes, but usually only single bones are found. Moreover, these finds often occur in archaeological contexts that provide limited information on cat ecology and relationship with people . Therefore, our knowledge on the behavior, habitat, and place in the human cultural landscape of ancient cats is still limited. Thus, rather than deliberate over individual isotopic traits, we exploited isotopic trends among broad groups of cats in relation to their chronology, geography, and socio-economic contexts to determine population-scale isotopic patterns.37     compared to the Baltic Sea region   , cats from rural sites differed too from those from other sites, except natural sites  locations.Among the sites located in the Baltic region, cats from strongholds, castles, rural, natural and urban sites were statistically similar to each other in terms of δr) Figs. . For δ1515N values to cats from other types of sites; except urban sites and castles. Their δ15N elevated values were attributed to higher contribution of marine food resources. For δ13C values, seaports statistically differed to strongholds, castles, and rural sites.Cats from seaports held a distinct position compared to other sites. These cats exhibited statistically significant different δ13C and δ15N , which was not detected in the Baltic region . The lowest δ13C and δ15N values were found at castle and rural sites, whereas the highest values related to seaports and a monastery located on the coast. The urban sites exhibited a wide range of values. The δ15N signal of single samples overlapped with the highest δ15N values detected at seaports, whereas the lowest signal fell in the range of castle and rural sites.For the North Sea region, isotopic values significantly differed between most site types, in terms of both δ5N Figs.  and S11.To detect geomorphological factors that might shape the isotope signal of cats, we grouped the sites into five categories: inland, river valley, lakeshore, sea coast, and lagoon/fjord Fig. ” section13C and δ15N values , with a weak similarity for inland vs. lakeshore pairs . This lack of differences was visible in terms of both δ13C and δ15N values  of a carnivore is controlled by many factors. These include biological factors  and indirect environmental factors, which are responsible for the isotopic signal in prey 53. For domestic carnivores, direct or indirect anthropogenic impacts might also play an important role in shaping isotopic signals. These include, but are not limited to, various human activities: (i) feeding on consumption/production waste or on selected food; (ii) creation of new landscapes, opening up habitats for new prey species; (iii) farming selected plants, which serve as a food base for herbivores ; and (iv) supplying food that would otherwise be of limited availability to a terrestrial carnivore (such as food acquired from fishing or distant trade). The feeding behavior of cats is also driven by local conditions, including currently available food resources, their relationship with people , and individual behavioral characteristics4. Recent studies of modern cat diet indicate high behavioral and feeding differentiation in domestic cats18.In general, the carbon and nitrogen isotopic signal (δ4 plants and location of the fishing grounds and the type of fish exploited) could enhance or obscure the isotope indicators of consumed resources.The most important factor shaping the isotope signal of the studied cats in the current study was the proportion of terrestrial and aquatic food resources in the diet. However, the regional isotope baseline (particularly the marine baseline), determined the degree to which differences could be detected through isotope values. In our dataset, the terrestrial and marine isotope signals were significantly different for cats from the North Sea region compared to those from the Baltic Sea region. Furthermore, the local background of human activity  cats had lower δ15N values  had similar isotopic patterns with high δes Figs. . Such elon Figs. . This is15N values and clearly lower minimum δ15N values  were statistically dissimilar from those on the sea coast. This result was due to slightly lower mean δ15N signal56, and omnivorous rodents, which forage on fishery wastes and exhibit elevated δ15N values in port towns58.The nitrogen and carbon isotopic signals in cats suggest an opportunistic shift toward local aquatic resources when available and a high reliance on such resources. This phenomenon might be related to scavenging fish remains or cats hunting terrestrial piscivores. Such prey might include sea birds, common in port towns and characterized by a marine-like δ13C values  vs. aquatic  resources. The carbon isotopic signal depends on marine  vs. non-marine  resources. The presence of C4 plants might also elevate the latter. The observed low δ15N and high δ13C variation in seaport cats indicated that aquatic resources were an important component of their diet, with mixed marine and non-marine resources, or mixed marine-littoral and marine-pelagic resources, or both.Cats from urban sites and seaports had the most varied δes Figs. a and 6. 15N, but also, highly varied δ15N values  at the basis of the trophic web. These C4 plants were possibly stored cereals, which served as a source of food for pest rodents, which, in turn, were preyed upon by cats. The presence of millet was confirmed in archaeobotanical material from Puck66. However, in the case of coastal sites, such as Puck, Kołobrzeg and Visby, the marine resources could also play an important role. Cats had significantly different δ15N values in these seaports versus Napole. These seaports had the highest, whereas Napole had the lowest values out of the entire dataset. This difference followed the seaport vs. rural site function, and likely reflected differences in the proportion of marine food available at each site.Detailed analysis of our dataset allowed us to detect some abnormalities in the isotopic pattern. For instance, several sites from the Baltic region  stood out from the whole dataset due to their high δnal Fig.  and S2. 13C values and moderate variability for δ15N values. The absolute δ13C values were similar to natural and rural sites, whereas δ15N values were usually higher , might have had limited access to fish and other water resources. The region has been densely forested until recent times, which suggests that the Logne cats could have had easy access to forest resources , reflected in their low δ15N and δ13C values.The castle of Logne was the only castle situated in the North Sea region in our database. Logne cats had a clearly lower δion Fig. . This re67. Marine fish, however, were commonly available in Bremen as evidenced by zooarchaeological studies and historical records. Some anadromous species  used to enter the Weser during their spawning season and might have been fished locally68. The Bremen fishermen were organized in a guild from at least 1489, which held the fishing rights down to the river mouth69. Additionally, Bremen was an important trade hub since at least the eleventh century, particularly involved in the stockfish and herring trade70.The opposite situation is visible in cats from Bremen, recording marine-like isotopic signals despite its location in the freshwater zone of the Weser13C values27, and available to synanthropic rodents  either directly from fields, storages in barns or granaries, or from processed products;Presence of cereal crops, cultivated in open fields, characterized by elevated δ4 cereal crops with high δ13C signals (such as millet or maize)71;Cultivation or import of C15N values in crops, especially in grains) and watering 72;Agricultural treatments, which may influence isotopic values of cultivated plants, such as manuring , and access to the meat of wild animals 74;Access to meat/dairy from domestic animals that forage on agriculturally treated or untreated food .Access to fish (and other marine resources), usually characterized by an elevated δIn general, domestic cats may follow three main foraging strategies: (i) hunting, (ii) scavenging, mainly on human waste, and iii) direct feeding by humans. Considering these strategies, we may define the following factors responsible for an isotopic signal of cat diet, synthesized in Fig. 61. Towns favor larger populations of synanthropic rodents, due to food stores and refuse. In addition, urban areas as trading spots provide various imported food resources, including marine fish, even in inland locations. However, even the largest inland towns cannot compete with seaports in terms of the availability of marine resources.In a human-dominated landscape, the isotopic signal of cat diet can be impacted by all of the listed factors. However, certain basic patterns related to variable anthropogenic and geographical settings can be predicted. In rural areas, crop pests are easy prey; however, the proximity of natural environments encourages the opportunity for cats to wander and feed on wild prey. Modern rural cats have a more diverse diet and a higher predation rate compared to those inhabiting urban sites1. Modern cats sharing the same environment, especially town-dwellers, exploit highly variable habitats and, hence, have highly varied trophic ecology72. Some cats may live as "backyard cats," , which are occasionally fattened by their human neighbors. Others might be "feral outcasts," which are the barely tolerated semi-wild dwellers of cellars, roofs, and hovels, living on their own and avoiding human contact. Still others might live as "indoor pets." Some cats even, exploit all these lifestyles.The ecology of the domestic cat is complex because it is an opportunistic exploiter that can maneuver among multiple available ecological nichesThe individualistic behavior and the taphonomic limitations mean that the isotopic signature of a single fossil cat specimen, or even of several specimens, might not represent the entire population. These limitations were revealed in our study through the high variability of the isotopic signal within the cats from some socio-economic types of sites  and geomorphological settings . However, our data also revealed relatively low isotopic variability in cats from specific locations . This phenomenon most likely relates to feral cats, which can permanently or temporarily adopt a wildcat-like lifestyle. Similarly to modern cat populations, the medieval cats tended to have more heterogenous diet in urban areas, as they consumed more anthropogenic food. At the same time, the predation rate was higher in rural and natural areas.The observed isotopic diversity means that stable isotope analysis could be a useful tool in elucidating the diet and lifestyle of individual cats. However, a local/regional isotopic baseline must always be carefully considered, as no global range may be arbitrarily attributed to the particular ecology of a cat. This study shows a promising perspective concerning the trophic ecology of fossil and recent cats, offered by an analysis of diet-related stable isotopes preserved in bones. Further investigations using sulfur stable isotope analysis and compound-specific carbon and nitrogen isotope analysis may deliver more in-depth insight into the domestic cat ecology.45 , were sampled and analyzed separately, but their isotopic values were averaged Dataset .Felis sp. Linnaeus, 1758) was carried out in the Royal Belgian Institute of Natural Sciences and the Institute of Archaeology, Nicolaus Copernicus University in Toruń, using reference collections of domestic cat and European wildcat skeletons. The bones were measured according to the methodology by A. von den Driesch75, using a digital caliper (0.1 mm reading error)  we adopted the discrimination method based on relative size using the log-ratio technique by T. P. O'Connor76  likely represent domestic cats due to their relatively small size, and have been regarded as such in further analyses. Specimens extracted from previous publications (n = 18) were also considered domestic cats.1. Thus, for our study we selected adult individuals (with bone fusion completed) and subadult individuals, i.e. older than weaning age, based on combined bone fusion or tooth eruption79. For fragmented bones, when it was not possible to apply the criteria above, we used overall bone size and stage of bone ossification as indicators of post-weaning age . In such cases, we used these years as the range end. In cases where chronology was provided in a descriptive way, we adopted the following scheme: late medieval = AD 1250–1500; post-medieval = AD 1500–1800.84);sea coast ;lakeshore ;river valley ;inland .We attributed a geomorphological context to the studied sites based on their geographic location Dataset . We usednatural ;rural ;urban ;70;seaport ; in this category, we also include Bremen, which was involved in marine fishing and trade in the pastcastle ;stronghold ;monastery .We attributed a socio-economic context to each site based on data from the published literature Dataset , also taFor cave sites, we provided the geographic coordinates of cave entrances. For open-air sites, we provided the coordinates of the center of a village/small town, center of a castle, or center of a street. We used Google Maps to obtain coordinates . To provide the distance from the sea, we used the "measure distance" option in Google Maps. We measured this as the shortest distance from the coordinate-collecting point to the nearest sea coast line (± 1 km).The bulk stable isotope analysis of carbon and nitrogen was performed on collagen extracted from bones. Dentine was not sampled to avoid bias from inter-tissue fractionation.85. Isotopic measurements (δ13C and δ15N) and elemental measurements  were performed in duplicate at the Stable Isotopes Laboratory at the Institute of Geological Sciences, Polish Academy of Sciences  using a Flash EA 1112HT elemental analyzer (Thermo Scientific) connected to a Delta V Advantage mass spectrometer (Thermo Scientific). Mean SEs were < 0.33‰ for δ13C and < 0.43‰ for δ15N values. We normalized all measurements to δ13C values of USGS40 and USGS41 standards and all δ15N values to the IAEA 600 standard. Details are provided in SI Appendix For samples with lab. no. CAT, collagen purification method followed the protocol after Krajcarz et al. and Bocherens et al.86, which was modified with an additional NaOH-wash between demineralization and hydrolyzation88. Collagen subsamples were sent to the Division of Soil and Water Management of the KU Leuven for stable isotope (δ13C and δ15N) and elemental  analyses. The atomic C:N ratio was then calculated. Analyses were carried out on a Thermo Flash HT/EA elemental analyzer, linked to a Thermo DeltaV Advantage Isotope Ratio Mass Spectrometer via a ConfloIV interface (Thermo Scientific). Data calibrations were done using IAEA-600 (caffeine) and two in-house standards (Leucine and muscle tissue of Pacific Tuna), which were previously calibrated versus certified standards. Analytical precision was typically better than 0.15‰ for both δ13C and δ15N values, this was based on the average of four replicates of the different standards in each batch of samples.. Details are provided in SI Appendix For all of the remaining samples, collagen extraction was carried out at the Royal Institute for Cultural Heritage (RICH) in Brussels. Samples were prepared according to the method of Longin13C or δ15N =  × 1000 in ‰ (parts per thousand), where R is 13C/12C or 15N/14N. The international references were Vienna Pee Dee Belemnite (VPDB) for carbon and atmospheric nitrogen (AIR) for nitrogen.Data were reported in delta (δ) notation: δF tests in the case of non-parametric distributions or unequal variances) to check for statistical differences between isotopic variances of selected groups of samples . The homogeneity of variance and normality were tested with Levene's tests (from means and medians) and a set of normality tests 89. Because in many cases the groups exhibited non-normal distribution and unequal variances, post-hoc pairwise comparisons were difficult to perform. Therefore, analysis of variance was run separately for each pair. In cases where post-hoc tests were possible for the entire set of pairs, we applied Tukey's test (after a significant ANOVA) with the Bonferroni correction of p values for multiple comparisons. All tests were performed with PAST software, v. 4.0590.We used one-way Analysis of Variance , sequencing on the Illumina platform, and processing of the sequencing reads were performed as in Baca et al.8, with the modification that only positions with a minimum of 3× coverage were called. We obtained mtDNA sequences for 57 specimens, which were used in phylogenetic analysis, along with 123 already published sequences10. The Maximum Likelihood Tree was generated using IQtree92. The implemented ModelFinder determined that GTR is the best-fit model and the branch support was assessed using ultrafast bootstrap method (SI Appendix Fig. Genetic analyses of 75 samples were performed at the Centre of New Technologies at the University of Warsaw, Poland. All samples were analyzed in facilities dedicated to working with ancient DNA following strict rules to avoid contamination. Samples were prepared and processed as described by Baca et al.Supplementary Information 1.Supplementary Information 2.


Electrochemical supercapacitors as an energy storage device have become trademark in current electronic, medical and industrial applications, as they are sources of impressive power output. Supercapacitors supply fast power output, suitable to cover the energy demand of future electronic devices. Electrode material design is a subject of intense research in the area of energy development and advancement, due to its essential role in the electrochemical process of charge storage and the cost of capacitors. The nano-dimensions allow for more electroactive sites, different pore size distributions, and a large specific surface area, making nanostructured electrode materials more promising. Electrode materials based on metal oxides, metal nitrides, and metal carbides are considered ideal for highly efficient electrochemical supercapacitors. Recently, much effort has been devoted to metal nitride-based electrodes and their diverse compositions as they possess higher electrical conductivity and better corrosion resistance, electrochemical stability, and chemical reactivity. Among these, titanium nitride (TiN), possesses high electrochemical stability, outstanding electrical conductivity, and a unique electronic structure. Nanocomposites based on titanium nitrides are known to deliver higher electrochemical performance than pristine nanostructured TiNs due to potential synergetic effects from both the materials. In this paper, recent advancements made in the field of nanostructural TiN electrode materials for SCs are reviewed along with their challenges and future opportunities. Additionally, some of the major techniques involved in the synthesis process are discussed, along with some basic concepts. However, because of their short life cycle and low power density, they are unsuitable for a variety of applications that require a large amount of energy in a short period [−1), elongated cycle life (>105), rapid charge–discharge processes and low cost. The device is promising in a wide range of applications ranging from hybrid electric vehicles, space crafts, memory back-up devices and portable electronic devices [3–5 × 10 4 S cm−1), good electrochemical stability, extreme mechanical strength, and low production cost [Uncontrolled use of fossil fuels not only leads to their rapid depletion but also raises substantial concerns about global warming and energy shortages. The progress made possible by the use of such energy sources is not said to be long-term and sustainable ,2,3. Hent period ,11. A su devices ,13. The  devices ,15. On t devices . Conduct devices ,18. Howe devices . Electro devices ,21. The  devices . These a devices . However devices ,25,26. A devices ,28. Howe devices ,30,31. T devices ,33,34. I devices . Althoug devices ,37. Thision cost . Interesion cost . The surion cost . Along wNanostructuring of electrode materials provides short ionic diffusion pathways and helps in increasing the specific capacitance ,42,43. TThe development of innovative and stable potential electrode materials relies on the synthesis techniques used. Different synthesis techniques are employed in the fabrication of nanostructured TiNs for SC electrodes, a few of which are briefly explained here.2 nanotubes and results in H-TiN nanopillars with a surface area of 23.1 m2g−1 and diameter under the range of 100–150 nm, with a length of 6 µm, as shown in −3 at a current density of 0.83 Acm−3. Both the large specific surface area and increased electroactive sites are attributed to the remarkable EC performance [−1 at a current density of 5 Ag−1, improving on the 107 Fg−1 for a pure TiN electrode. Additionally, for a long cyclic process , the TiN@C electrode retained 91.7% of its initial capacitance [−3. The morphology and the synthesis process of TiN nanowires is shown in −1 at a 10 mV s−1 of scan rate. Additionally, high capacitance retention of 80% was achieved after running 10,000 charge–discharge cycles. The carbon coating is attributed to the enhanced performance of PVA–KOH gel electrolyte-based flexible SC [−1 at 2 Ag−1 of current density. The synergetic contribution from both the counterparts is attributed to the remarkable performance [One of the most often used methods for fabricating nanostructured SC electrodes is the solution-based hydrothermal synthesis method, which is simple and environmentally benign . This meformance . To provacitance . Liu et xible SC . The eleformance . The abo−3 mbar–10−2 mbar), temperature and voltage, results in the formation of defect-free thin films [−1 at 1 Ag−1 with awesome capacitance retention of 92.6% for a long number of cycles of 30,000 with a remarkable energy density of 26.28 Wh kg−1 and power density of 12.9 kW kg−1. The nanopyramid configuration is attributed to the enhanced performance [−3 with negligible decay of capacitance over 20,000 cycles. The performance is attributed to the doping of nitrogen into TiO2 layers, porosity and thickness of the deposited film [−2 is delivered in the presence of 1M aqueous KOH electrolyte. Moreover, the TiVN thin film-based electrode showed negligible capacitance decay over 10,000 cycles. A synergetic contribution from both the counterparts attribute to such good capacitance retention [−1 at 5 mV s−1 of scan rate with good energy and power densities [Magnetron sputtering has evolved to become an advanced technique for the deposition of highly uniform and pure thin films of different metal oxides, nitrides and sulfides . The tecin films ,59,60. Tin films . Magnetrformance . Recentlted film . In anotetention . The conetention . In thisensities .−2 at a high scan rate of 100 mV s−1 [2–TiN nanotube arrays through the facile electrodeposition process has been reported by Dang et al. The TiN nanotubes were synthesized using anodization of Ti foil using ammonia. The 1D MnO2–TiN nanocomposite showed an improved specific capacitance of 681 Fg−1 at a high current density of 2 Ag−1 with only 3% capacitance decay after 1000 continuous cycles. The high conductivity of TiN nanotubes and redox active behavior of MnO2 contributed significantly to the EC performance [2N), high electric conductivity (0.2 S cm−1), enriched electrochemical stability and high charge storing ability. Xie et al. tried to incorporate MoNx with TiN as MoNx–TiN NTA with the help of electrodeposition process followed by nitration process in ammonia. The average diameter of the nanotubes was found to be 110–130 nm with a length of around 4 µm , NaF and NaCl are utilized as raw materials. The authors used molar ratios of the Ti and graphite of 1:1, 1:2, 1:3 and 1:4, whereas the NaCl and NaF weight ratio was 10:1. Firstly NaF and NaCl were added equally to the Ti and graphite and were then mixed in an alumina crucible used to place the powdered mixture and maintain the temperature at 1100 to 1400 °C for 3 hours in an argon atmosphere . The forat 1 atm .6 S m−1), cubic crystal structure, high melting point of 3050 °C and density ~5.40 g cm−3 [−3 with an energy density of 0.34 mWh cm−3 at a current density of 0.05 A cm−3. Moreover, remarkable capacitance retention of 136% was achieved after running 20,000 charge–discharge cycles. The 3D flower-like morphology, high conductivity and good electrochemical stability are attributed as reasons for the remarkable EC performance [−2 with negligible capacitance loss after 10,000 cycles [2g−1 using a two-step halide approach. The different nanostructures were obtained by varying the heat treatment temperatures and were evaluated for their EC performances [Nanostructured titanium nitride is well known for its electronic conductivity comparable to that of metals  but suffer from poor electronic conductivity and low specific surface area. One of the strategies to improve the electric conductivity of metal oxides is to incorporate them with conducting TiNs as a conducting substrate or as a composite material [x NTAs over highly conducting TiN cores to assemble MoOx–TiN–MoOx as a composite electrode, one that comprises the high conductivity and pseudocapacitance of a TiN core with the pseudocapacitance arising from a TiN shell, as well as the electroactive sites. This in turn improved the capacitance and cycling performance, with the specific capacitance delivery of 97 mF cm−2 at a scan rate of 1 mA cm−2. The electrochemical stability of MoOx contributed significantly to the symmetrical SC device, showing a volumetric capacitance of 24 Fcm−3 and the device exhibited the full capacitance retention over 10,000 cycles. The dual-layered MoOx NTA over the thin TiN provided high mass loading with a large surface area and was attributed as the reason for the increase in the device’s overall performance [−1 at a scan rate of 2 mV s−1 due to their fibrous structure that provided a large area of access to the electrolyte ions. The electrode maintained a capacitance retention of 88% over 500 cycles. The improved specific capacitance delivery and good rate capability were ascribed to the presence of TiN and VN in the composite [2SO4 as electrolyte. The magnetron sputtered grown bimetallic nitride TiNbN as binder-free electrode and delivered a capacitance of 59.3 mF cm−2 at a scan rate of 1 mA cm−2 with outstanding cyclability after 20,000 cycles. The asymmetric configuration led to an extended voltage window of 1.6 V with enhanced energy and power densities as 74.9 mWh cm−3 and 8.8 W cm−3 respectively. The synergetic contribution from Ti and Nb were ascribed as reason for the boost in EC performance [Due to the excellent electrical conductivity and electrochemical stability, nanostructured TiNs provide a conducting framework to hold various transition metal oxides (TMOs) such as MoO2O4 etc. ,42. The material . Peng etformance . Fibers formance . Incorpoomposite . The pooformance . Althoug−1). Among these, TiNs have attracted more attention due to their higher conductivity, which is closer to pure metal. For example, Lu et al. have reported free-standing TiN nanowires grown on a carbon cloth using a two-step synthesis method. Firstly, they uniformly grew 100 to 200 nm TiO2 nanowires on carbon cloth via a seed-assisted hydrothermal synthesis method. After that, these nanowires were thermally annealed in a NH3 atmosphere and with a constant temperature in the range of 700–1000 °C to obtain the TiN. The fabricated flexible free-standing TiN electrode shows an excellent electrochemical performance. In this work the authors claim that the flexible solid-state TiN SC retained 83% retention after 15,000 cycles. The electrochemical performance of the flexible solid-state TiN SC could open up new opportunities for a flexible solid-state SC based on TiN and its composites [Flexible energy storage devices are fundamental to the development of next generation wearable, compact and portable electronics for medical, military and civil applications such as health tracking devices, computers, television, or flexible displays on phones. Flexible supercapacitor devices are highly attractive in comparison with batteries as they combine the inherent high power density (≥10 kW–kg), fast charging–discharging capability, longer life cycles and good mechanical flexibility. In conventional supercapacitors they consist of the outer case, the current collectors in the form of the metal foils, and cathode and anode electrodes in the electrolyte separated by the ion transport layer. In the case of flexible supercapacitors, the highly conducting and flexible carbon network serves as both the electrodes and current collector. Therefore, the architecture of the flexible supercapacitors is highly conducting and lightweight and is further simplified for portable electronic devices. Thus, self-supported and flexible electrodes have been paid intensive attention. Deposition of the active material on the flexible textile is one of the most effective and widely reported methods to fabricate flexible free-standing electrodes. Recently, highly conductive transition metal nitrides such as TiN, VN, MoN, FeN have been used in flexible SCs due to their high capacitance (100–1340 Fgmposites . Recentlmposites . Table 1To increase the performance of supercapacitors (SCs) enough to power advanced electronics and other technologies, the development of innovative and promising electrodes is the most pressing need. Recent research carried out in the development of SCs has emphasized the nanostructuring of electrode materials. These changes are vital due to the following facts: (1) a smaller particle size with sub-nanoscale dimensions provides a large specific surface area for the generation of electrons and the accumulation of ions on the electrode surface; (2) nano-dimensional architecture facilitates short paths for the diffusion of electrolyte ions, resulting in higher wettability of electrode surface; (3) nanostructuring of electrode materials strengthens the whole electrode, which helps in providing electrochemical stability; (4) fabrication of SC electrodes with a wide range of pore-size distributions with various dimensions are only possible by nanostructuring, and is therefore very important for accessing the maximum surface area of electrodes by electrolytes; and (5) nanostructuring provides high surface-to-volume ratio to electrodes, which increases their capacitance. Along with the strategy of nanostructuring electrodes, choice and optimization of an electrode material is equally important. Among several types of electrode materials, transition metal nitrides are of huge importance due to their high conductivity, structural stability and remarkable electrochemical performance. Titanium nitride (TiN) has recently been explored for potential use in SC electrodes. The material is well known for its excellent electronic conductivity, comparable to that of metals, and its good mechanical strength. The nanostructures of TiNs add value to their novel characteristics in terms of higher specific energy, specific power, cyclic stability and large operating potential window. Considering the promising features of nanostructural TiNs, it is important to summarize the latest advancements made in nanostructural TiNs, with the help of different synthesis techniques, incorporating their importance, advantages and complications. In this review, a comprehensive overview of SC performance based on nanostructural TiNs was provided, including a brief introduction of some techniques involved in the fabrication of electrodes. The EC performance of pristine nanostructured TiNs can be enhanced by incorporating these with other materials such as carbonaceous materials, metal oxides, metal sulfides and conducting polymers. The composite hybrid form of nanostructural TiNs incorporates the synergetic effect of all the counterparts, resulting in extraordinary conductivity, electrochemical stability and mechanical flexibility. The concept of free-standing and binder-free nanostructural TiNs or composites avoids the usage of any binder or conducting agent and provides ultra-long cycling performance to the electrodes. However, in comparison with other SC electrode materials, TiN has received little attention, and, as a result, research on this material is limited. The lack of understanding of the original and fundamental mechanism of charge store behavior in the crystal lattice is the cause of this scarcity. The process of their capacitance decay is similarly poorly understood. As a result, there are numerous research gaps in existing nanostructured TiNs that need be investigated in order to increase the EC performance of SCs. Apart from the obvious advantages of TiNs, there are also some intrinsic drawbacks limiting their practical applications. There is a challenge in large-scale controllable synthesis. Fabrication of TiNs with controlled/specific structures and crystal planes usually requires multistep processes and high temperature treatment, which make the process costly, complicated, or even uneconomical.