Most public cord blood banking programs are currently facing financial difficulties due to a progressive decline in the number of cord blood transplants performed worldwide and to a high discard rate of the donated units caused by progressively increasing thresholds of the stem cell dose required to perform safe and effective hemopoietic cord blood transplants. Recycling a proportion of unused cord blood units to prepare novel cord blood components obtained with minimal manipulation  and to develop more technologically complex products regulated in the US as Cellular and Gene Therapy Products and in Europe as Advanced Therapy Medicinal Products [e.g. virus-specific T cells (VST), natural killer (NK) cells, induced pluripotent stem cells (iPSCs) is a promising strategy to increase the therapeutic value and reduce the financial deficits of public cord blood banking. Based on encouraging preliminary evidences reported in the literature, additional laboratory studies, large multicenter clinical trials and international regulatory harmonization are necessary to achieve these important goals. This article describes organizational, methodological and regulatory advancements developed in Italy and Spain to promote the clinical use of cord blood platelets, plasma and red blood cells. During the last 3 decades, newborn’s blood remaining in the placenta after term deliveries has been collected from the umbilical cord, processed and cryopreserved in public cord blood (CB) banking programs implemented following the first successful hemopoietic CB transplants pioneered by Eliane Gluckman, Hal Broxmeyer and Joanne Kurtzberg in the late 1980s. So far, a global cryopreserved CB inventory of about 800,000 units has allowed the performance of more than 40,000 transplants in pediatric and adult patients affected by blood cancers, immune deficiencies, metabolic disorders and hemoglobinopathies .+ cells, the latter two used as surrogate markers of the unit’s hemopoietic stem cell content. More recently, multiple studies have conclusively shown that the clinical outcome of CB transplant is positively associated with the hemopoietic stem cells administered dose. This evidence led to the identification of a progressively increasing threshold of the minimum TNC and CD34+ cell count required before starting expensive procedures for banking and long term cryopreservation, which currently can determine disposal rates of the collected CB units as high as 90%  and total CD34h as 90% . This hih as 90% , which fh as 90%  and noveh as 90% .+ cell counts as its inputs) ; B: unit banking rate equal to 20% , mean TNC = 12.4 × 108 and CD34+ cells = 4.3 × 106; C: unit banking rate equal to 6% , mean TNC = 17.9 × 108 and CD34+ cells = 6.7 × 106; and D: unit banking rate equal to 2% , mean TNC = 24.8 × 108 and CD34+ cells = 11.1 × 106. Scenarios A and B, with the largest proportions of banked units, provided similar numbers of transplants (284 and 269 respectively), but unsustainable economic deficits of −5,886,986 and −3,046,101 USD respectively. Scenario D, with the lowest proportion of banked units, was associated with lower deficit , but this was associated with a significant reduction of the bank’s therapeutic value, i.e. the number of units distributed for transplant (n = 143). The authors considered scenario C as the most economically sustainable and therapeutically appropriate as it provided 219 transplants and a significantly lower deficit of −976,160 USD and concluded that “a pre-freezing level of 18 × 108 TNC would be a cost-effective strategy to deliver therapeutic value to patients with a minimum financial deficit for the bank”  and “utilization score” (as determined with a formula using TNC and CD34 inputs) . The authe bank” .+ cells, volume, red blood cell count per microliter and age (hours) at reception. These authors identified five scenarios  and determined that CB units graded A+  accounted for 5% of total expenditures while providing 29% of all shipped grafts (65/227), as opposed to units graded D , which accounted for 37% of processing cost despite being the source of only 11% of shipped transplants (24/227).A “utilization-based” unit selection procedure using the formula originally developed by Magalon et al.  was repoin utero and ex utero collection of a single CB unit  can find a match to undergo an allogeneic transplant.In Italy, CB banks (CBBs) are public hospital-based health facilities that collect, store, and distribute CB hematopoietic stem cells (HSC) on behalf of the National Health Service. CB banking, which was started in 1993, was formally organized into the Italian Cord Blood Network (ITCBN) in 2009, when a decree of the Ministry of Health assigned to the National Blood Center  the coordination of the CB banking network in cooperation with the National Transplant Center  . The fun+ cells. In July 2011, the ITCBN set a threshold of 12×108 TNC pre-processing for allogeneic unrelated units, which was increased in 2016 to 16 × 108 TNC pre-processing and 12 × 108 TNC with 2 × 106 CD34+ cells for banking. Up to December 2020, the total ITCBN inventory included 41,831 allogeneic unrelated units, 1,581 of which have been distributed to national and international transplant centers.Eighteen ITCBN facilities are currently operating in 13 Italian regions, in compliance with national regulations , guideliThe CBBs belonging to the ITCBN are an integral part of the Regional Health Services, which are responsible for the organization and administration of publicly financed health care, including the cost of public CB banking. The reimbursement fee for one allogeneic unrelated CB unit amounts to 17,000 € . An ITCBItalian law no. 219 “New discipline of transfusion medicine activities and national production of blood derivatives” of 21 October 2005 includes CB donation and collection within blood transfusion activities . CB bankIn 2014, considering the large availability of allogeneic CB units not suitable for transplant due to low HSC content, the ITCBN, under the coordination of the CNS, launched a national programme to standardise the preparation of a new CB component, allogeneic cord blood platelet concentrate (CBCP) suitable for the preparation of CB platelet gel (CBPG) . This prIn parallel, thanks to the collected scientific evidences, CB platelet concentrate, platelet gel and eye drops have been included in the transfusion technical norm, specifically the Decree of the Ministry of Health of 2 November 2015, integrated by the Decree of 1 August 2019 . The latIn the current worldwide public CB banking scenario, Italy is promoting a new hub-and-spoke ITCBN model. While a centralization of CB storage for transplant purposes in a small number of cryogenic facilities will improve the economic sustainability, maintenance of a large number of collection facilities will help increasing the HLA diversity of the national inventory. In a larger number of CB banks, preferably located in the transfusion services, a systematic multicomponent production could be implemented from a proportion of CB units unsuitable for HSC transplant. A regular production of the novel CB components might open the door to a regulated partnership with industrial stakeholders interested in manufacturing clinical grade biological drugs (e.g. eye drops).8 TNC, including a stock renewal of units not meeting this TNC count threshold.- To meet the national CB inventory target of 60,000 CB units with at least 8 × 10- To implement common quality criteria of the Spanish CB inventory.- To develop research programs on new therapeutic uses of CB.- To communicate to the society the benefit of public CB banking with regard to both the consolidate uses or the new potential applications.- To design a common training and dissemination program on good clinical and manufacturing practices for health care professionals and regulatory bodies.In Spain six public CB banks are in operation. These banks follow a national guide   aimed at8TNC, 2 × 106 CD34+ cells, with a cell viability above 85%. This recommendation implies that units accepted for processing for transplantation should contain at least 15 × 108 TNC and 4 × 106 CD34+ cells. This very high threshold means that less than 20% of collected CB units will be further processed for transplantation and a substantial amount of collected unit will be wasted.Following this guide, the Spanish CB network agreed that new units listed by REDMO should have a minimum post-processing count of 9 × 10On December 31st, 2020, the Spanish CB inventory included 64,638 units, 77% of them meeting the agreed quality thresholds. Therefore, there is room for improving the inventory and CB banks are continuing collecting units. To increase this number to the desired target, the 6 CB programs have collected a median of 7142 (2365-13081) units per year during 2016–2020 . From all collected units, only 13%  have finThe first CB unit available through REDMO was registered in april 1997 and the first shipment occurred on 5 May 1997, a unit sent to United States. Since then a total of 74,490 units have been transferred to REDMO and 86.7% of them are still available for clinical transplantation. During this time a total of 3,515 units were shipped for transplantation and 8.5% were deleted from the inventory due to various reasons (the most frequent (75%) after quality review during stability or reservation studies). The utilization rate in Spain is therefore 4.7% of the total registered inventory.To estimate the cost of the national inventory, the data provided by Arrojo et al. , who estFollowing the ratio of use decreased by 20% in the last 5 years and the re-orientation of PNSCU requiring researching in new applications, Barcelona CBB and the program Concordia BST, including the banking activity of six donation programmes in six autonomous regions of Spain plus Andorra, started the development of new therapeutic products from units not suitable for transplantation. In this regard a processing method to generate a new product based on CB platelets and plasma was validated . This neBlood donation and transfusion are subject to extensive regulation based on documents with different rank and scope at European and national level. In Spain, Royal Decree 1088/2005  establisA Member State may, in accordance with legislation in force and to fulfil special needs, exclude from the provisions of this Directive medicinal products supplied in response to a bona fide unsolicited order, formulated in accordance with the specifications of an authorized health care professional and for use by his individual patients on his direct personal responsibility”  published a resolution establishing that autologous plasma and its fractions, components or derivatives are “medicinal products” but they fall under a separate category as they are used “to meet special needs” . This reibility” , the AEMnon-substitutive therapeutic use of any other product based on non-autologous plasma would receive the treatment of biological medicine capable of being industrially produced and its authorization would be regulated by Royal Decree 1345/2007, of October 11, which regulates the procedure of authorization, registration and dispensing conditions of industrially manufactured medicines for human use, so subject to the pharmaceutical regulations  and an eye drop formula from cord blood platelet lysate (CBED) have been approved by AEMPS to be used in clinical trials. CBED was also employed in a programme of compassionate use in 33 patients (46 eyes) unresponsive to conventional treatments who required urgent intervention. Promptly available CBED resulted in a well-tolerated allogeneic treatment that showed evidence of efficacy . The reiThe different regulatory status of the same CB components in Italy (as blood components) and Spain  indicates the need of regulatory harmonization. The current EU Directive on Blood  is underMulticomponent CB Banking using platelets, plasma and red blood cells from a proportion of currently discarded CB units could contribute to increasing the therapeutic value and reducing the financial deficit of public CB banking programs . Interna


The metabolic syndrome (MetS) is correlated with disorders of the reproductive system, such as the polycystic ovary syndrome (PCOS). While consumption of a diet rich in carbohydrates is linked to the development of MetS, it is still unclear if this diet leads to ovarian dysfunction and PCOS.We investigated the influence of a high-sucrose diet (HSD) on the ovarian milieu of Wistar rats and studied the correlation between high consumption of sugary drinks and the prevalence of PCOS in women.Wistar rats were given a standard laboratory diet  or HSD  from postnatal day 21 to 120. Animals were evaluated weekly to calculate food intake, feed efficiency and weight gain. Both onset of puberty and estrous cycle were monitored. Metabolic serum biochemistry, organ morphometry and ovarian histology were performed upon euthanasia. In parallel, a fixed-effects multiple linear regression analysis was performed using data from Brazilian states (459 state-year observations) to test the correlation between the consumption of sugar-sweetened beverages (surrogate for HSD intake) and the prevalence of PCOS (surrogate for ovarian dysfunction).HSD animals showed increased adipose tissue accumulation, hyperglycaemia and insulin resistance when compared to CTR. Interestingly HSD rats also entered puberty earlier than CTR. Moreover, ovaries from HSD animals had an increased number of atretic antral follicles and cystic follicles, which were correlated with the hypertrophy of periovarian adipocytes. Finally, there was a positive correlation between the intake of sugary drinks and prevalence of PCOS in women of reproductive age.HSD ingestion leads to ovarian dysfunction in rats and could be correlated with PCOS in women, suggesting these alterations could lead to public health issues. Therefore, we reinforce the deleterious impact of HSD to the ovarian system and suggest that the reduction of added sugars intake could be beneficial to ovarian health. Metabolic syndrome (MetS) is defined as the presence of at least three of the following: obesity, insulin resistance (IR), dyslipidaemia, hypertension and increased fasting glycaemia . MetS prThe link between MetS and reproductive disorders is multifaceted due to hormonal imbalances. For instance, it has been reported that hyperinsulinemia and IR are involved with hyperandrogenism, since insulin stimulates the production of ovarian androgens in synergy with luteinizing hormone . Both hyIn this regard, the effects of high-fat diet (HFD) consumption have been studied in rodents. HFD ingestion is reported to cause irregular estrous cycle, decreased fertility rates and impaired follicular development, while increasing ovarian inflammation and leptin levels . MoreoveTherefore, taking into account the high prevalence of obesity amongst women of reproductive age and the scarcity of studies in rodents and humans that explore the effects of high-sucrose diet (HSD) intake to ovarian function, we hypothesized that HSD ingestion leads to ovarian dysfunction. Of note, the HSD used in this study mimics dietary intake of sucrose found in Latin American countries . Our maiad libitum access to water and food. All experimental procedures were performed in accordance with the rules of Brazilian Council for the Control of Animal Experimentation and approved by the Ethical Committee on Animal Use and Welfare at UFMA, ruling number 23115.007440/2016-71.Female Wistar rats (Rattus norvegicus) with 21 days of age and approximately 45 g were used, supplied by the Animal Facility House of the Federal University of Maranhão (UFMA) and kept in the Bioresources Unit of the Experimental Physiology Laboratory. Animals were kept at constant temperature (23 ± 2°C), 12h light/dark cycle and qsp, as previously described (HSD was manufactured with powdered standard chow (40%), condensed milk (40%), refined sugar (8.5%) and filtered water escribed  for a fiWeaned female Wistar rats were provided by the Animal Facility House of UFMA and randomized into two groups: standard laboratory diet  and HSD (n = 8) groups. Dietary intervention started at postnatal day (pnd) 21 and continued until euthanasia, at pnd 120. Animals were weekly monitored for: weight gain, food intake and feed efficiency. In order to verify the onset of puberty, the vaginal opening was observed daily, starting at pnd 21. From the pnd 90 until euthanasia, the phases of the estrous cycle were determined as described below. During the last week of dietary intervention, animals were submitted to glucose and insulin tolerance tests. The Lee Index ((body weight (g)1/3 ÷ naso-anal length (cm)) × 1000) was used to assess body mass index  at pnd 1In order to determine the beginning of sexual maturity, the vaginal opening was observed daily, beginning at pnd 21. The estrous cycle was evaluated daily starting at pnd 90 until pnd 120, between 4:00 pm and 6:00 pm, according to Marcondes et al.  at 0 (before glucose administration), 15, 30, 60 and 120 minutes after glucose administration. For ITT, fed animals received recombinant human insulin (2 IU/kg preheated to 36 °C). Glycaemia was measured at 0 (before insulin administration), 3, 5, 10, 15 and 20 minutes after insulin administration. The constant rate of glucose disappearance (kITT) was calculated from the regression slope obtained from the log-transformed glucose values between 3 and 20 minutes after insulin injection  was administered by oral gavage. Blood was collected through puncture of the caudal vein and glycaemia assessed using a glucometer (Accu-Chek Activenjection .g for 10 min) to obtain serum. Fasting triglyceride and cholesterol levels were detected in serum using spectrophotometric test kits following the manufacturer’s guidelines . Fasting and fed blood glucose levels were defined as time 0 of GTT and ITT assays, respectively. The TyG index was used to infer insulin resistance using the formula: Ln [glucose (mg/dL) x triglycerides (mg/dL)/2] /2] .Rats at the oestrous phase were euthanized and the right ovary and surrounding fat pads were removed, cleaned, fixed in 4% paraformaldehyde for 24 hours and kept in 70% ethanol. The right ovary was immersed in paraffin, cut into 6 µm slices and stained with hematoxylin-eosin (HE). Since an oocyte has a diameter of 20 to 30 µm, we analysed one section every six cuts, therefore ensuring a 36 µm distance to prevent us from double-counting the same follicles. On average, nine slices in three sections were analysed per ovary. Full details on the classification of follicles, definition of atresia and follicular cysts can be found in previous reports of our lab , 24. RepThe fat pads surrounding the right ovary were cut into 6 µm sections and stained with HE. Histology of adipocytes was performed as described previously . At leasData were collected from the Global Health Data Exchange, the Institute for Health Metrics and Evaluation, the Brazilian Institute of Geography and Statistics (IBGE) and the Brazilian Ministry of Health. Data were acquired for the 26 states and the Federal District from 2000 to 2017, totalling 459 state-year observations. The dependent variable was the prevalence of PCOS per 100,000 habitants, while the main independent variable was the summary exposure value (SEV) for diet high in sugar-sweetened beverages, as a surrogate for high sucrose intake. Control variables consisted of gross domestic product (GDP) in Brazilian Reais (R$) per capita, SEV for high LDL-cholesterol and SEV for high fasting glycaemia. Prevalence and SEV data were collected for women at reproductive age (15 to 49 years) and were defined and calculated by the global burden of diseases (GBD) study group  for the ecologic study. For the rodent study data were analysed using unpaired Student’s t test or two-way ANOVA, with Tukey’s post-test and n=6-8, except for ITT (n=5) and the histology of periovarian fat (n=4). The sample size for the animal study was calculated using the free software for sample calculation: G*Power 3.1 , Germany). The sample size of eight animals per group was found by inserting in the software sample power of 80% (1 − β = 0·8), significant level of 5% (α = 0·05), and parameters from previous studies on the effects of MSG on the number of ovarian cysts, antral follicles, and adipocytes area , 24. ForTo assess the impacts of HSD on the development of obesity, animals received HSD or CTR diet for a period of 14 weeks, from pnd 21 to 120. Body weight was assessed twice a week and fat pads were isolated at the end of the dietary intervention. As shown in vs HSD 102.30 ± 4.43, p = 0.03) and fed  blood glucose when compared to CTR rats , suggesting these animals presented IR , cholesterol and triglycerides were measured. In addition, OGTT and ITT were performed to assess glucose tolerance and insulin sensitivity, respectively. As shown in vs HSD 40.25 ± 0.16, p <0.0001), suggesting that HSD ingestion led to early puberty. In spite of earlier vaginal opening, HSD rats ovulated and cycled normally, similar to CTR animals  and oestrous cycle were monitored Figure 3vs HSD 1.22 ± 0.28, p = 0.002) and cystic follicles  when compared to ovaries from CTR rats. The number of corpora lutea was also unaltered in HSD rats when compared to those of CTR animals   and the prevalence of PCOS (surrogate for ovarian dysfunction) in women at reproductive age. Controls for income and other risk factors, namely hyperglycaemia and dyslipidaemia were used, since similar alterations were seen in HSD rats. The prevalence of PCOS in women at reproductive age was positively correlated with the consumption of sugar-sweetened beverages  and the prevalence of PCOS (proxy for ovarian dysfunction) in the Brazilian population. It was evident that there was a positive correlation between consumption of sugar-sweetened beverages and prevalence of PCOS. Interestingly, there was a lag effect between the consumption of sugar-sweetened beverages at 25-29 years and the prevalence of PCOS at 35-39 years that was lost when an older bracket of the population was analysed. This is in line with the programming effect of HSD consumption at younger ages, such as demonstrated for HSD rats. In parallel, it has been reported that adolescents with PCOS tend to consume a large amount of foods rich in sugar , while, in vivo using a larger sample size. Third, we were unable to ascertain if HSD rats developed PCOS due to lack of hormonal levels and we suggest this to be explored in the future. Finally, the ecologic study does not allow causal inference between the consumption of sugary drinks and the prevalence of PCOS. While other factors could influence the prevalence of PCOS, controls for income and fixed effects models were employed to account for some externalities that could influence the prevalence of PCOS. Nonetheless, it is imperative to address in the future if HSD can lead to PCOS in women.Some limitations of the current study need to be addressed in the future. First, while we identified early puberty in HSD rats, we were unable to explore mechanisms that led to this event. A feasible hypothesis is that HSD could cause the hypertrophy of periovarian fat in prepubertal rats, as discussed above. Future studies should assess if periovarian fat pads are altered in prepubertal rodents exposed to HSD. Second, further studies are needed to extend our observations In conclusion, data herein presented show that early exposure to HSD causes metabolic and ovarian dysfunctions in rats, which could have implications to the prevalence of PCOS in women. More studies are needed to address the link between HSD consumption and PCOS development. Moreover, our study suggests that early introduction of added sugars in the diet, particularly following breastfeeding cessation, is detrimental to female reproductive health. Therefore, sucrose-limiting policies could potentially hinder the deleterious impacts described above.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval were not required for the study on human participants, because data were collected from a public database, in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by the Ethical Committee on Animal Use and Welfare at UFMA, ruling number 23115.007440/2016-71.GM, TC, RB, and CV have performed experiments and analysed data. JS has analyzed data and written the manuscript. AP has supervised experiments, conceived the study, analyzed data, and reviewed the manuscript. RG has performed experiments, conceived the study, analyzed data, and written the manuscript. All authors contributed to the article and approved the submitted version.This work received financial support from the University of Reading, Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico, Tecnológico e Inovação do Estado do Maranhão – FAPEMA (IECT 05593/18) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. GM received a PIBIC fellowship from FAPEMA. JS, TC, RB, and CV received a Masters fellowship from CAPES. AP received a Researcher fellowship from CNPq (308163/2019-2).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.


Anabaena variabilis is a filamentous cyanobacterium that is capable to differentiate specialized cells, the heterocysts and akinetes, to survive under different stress conditions. Under nitrogen limited condition, heterocysts provide the filament with nitrogen by fixing N2. Akinetes are spore-like dormant cells that allow survival during adverse environmental conditions. Both cell types are characterized by the presence of a thick multilayered envelope, including a glycolipid layer. While in the heterocyst this glycolipid layer is required for the maintenance of a microoxic environment and nitrogen fixation, its function in akinetes is completely unknown. Therefore, we constructed a mutant deficient in glycolipid synthesis and investigated the performance of heterocysts and akinetes in that mutant strain. We chose to delete the gene Ava_2595, which is homolog to the known hglB gene, encoding a putative polyketide synthase previously shown to be involved in heterocyst glycolipid synthesis in Anabaena sp. PCC 7120, a species which does not form akinetes. Under the respective conditions, the Ava_2595 null mutant strain formed aberrant heterocysts and akinete-like cells, in which the specific glycolipid layers were absent. This confirmed firstly that both cell types use a glycolipid of identical chemical composition in their special envelopes and, secondly, that HglB is essential for glycolipid synthesis in both types of differentiated cells. As a consequence, the mutant was not able to fix N2 and to grow under diazotrophic conditions. Furthermore, the akinetes lacking the glycolipids showed a severely reduced tolerance to stress conditions, but could germinate normally under standard conditions. This demonstrates the importance of the glycolipid layer for the ability of akinetes as spore-like dormant cells to withstand freezing, desiccation, oxidative stress and attack by lytic enzymes. Our study established the dual role of the glycolipid layer in fulfilling different functions in the evolutionary-related specialized cells of cyanobacteria. It also indicates the existence of a common pathway involving HglB for the synthesis of glycolipids in heterocysts and akinetes. Anabaena variabilis ATCC 29413 can undergo a variety of cellular differentiation processes forming motile hormogonia, nitrogen-fixing heterocysts, and spore-like dormant cells called akinetes, and therefore, serves as a model organism to study the cell differentiation process in prokaryotes  fixing heterocysts appear in a semi-regular pattern along the filament in response to insufficient supply of combined nitrogen under aerobic conditions , reduction of metabolic activities and formation of a thick multilayered envelope , 2018. Talr5351–alr5357  in Anabaena sp. PCC 7120  domain and a C-terminal thioester reductase (TER) domain  domain . The chaPCC 7120  demonstrAnabaena variabilis ATCC 29413 strain FD (–2 s–1) at 28°C with shaking at 120 rpm in standard medium of 3. The solid media remained undiluted with 1.5% (w/v) Difco Agar. For growing the mutant strain, 50 μg ml–1 neomycin was added to the medium. To induce heterocyst differentiation, the exponentially growing cultures (ODnm750  0.4–0.5) were harvested and washed three times in nitrate-free AA/4 medium, resuspended in same medium equal to the original volume, and cultivated under nitrogen depleted conditions.Vegetative cultures of train FD  and deritrain FD  were groEscherichia coli strains were grown in lysogeny broth (LB) medium at 37°C, supplemented with the following antibiotics: 50 μg ml–1 kanamycin (Km), 25 μg ml–1 chloramphenicol (Cm), 25 μg ml–1 streptomycin (Sm) and 100 μg ml–1 spectinomycin (Sp), when required. For growth on solid medium, 1.5% (w/v) agar was added. The E. coli strain Top 10 was used as a host for plasmid constructions. For triparental conjugation, the E. coli strain J53 (bearing the conjugative plasmid RP4), strain HB101 (bearing the helper plasmid pRL528 and the cargo plasmid), and the wild type (WT) A. variabilis culture were used  was inactivated by insertion of the neomycin-resistance-conferring cassette (C.K3) into the genome by double-crossover homologous recombination  were induced to differentiate akinetes by transferring them to low light condition (2–3 μmol photons m–2 s–1) with shaking at 50 rpm ] was added to the cell suspension (in a ratio of 1:100) and incubated at room temperature (RT) for 5–10 min.To observe the heterocyst envelope polysaccharide layer, Alcian blue staining was performed . Alcian The culture containing heterocysts was mixed with the TTC solution [0.05% of TTC (w/v) in the final mixture] and incubated in dark for 15–30 min at RT .In order to visualize the glycolipid layer in the heterocyst and akinete envelope, samples with heterocyst and akinetes were stained with boron-dipyrromethene difluoride (BODIPY) 493/503  as described previously . After aFor electron microscopy studies, individual cultures containing akinetes or heterocysts were fixed with 2.5% glutaraldehyde and post-fixed with 2% potassium permanganate followed by immobilization in agarose. After dehydration by successive increment of the ethanol concentration, the samples were embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate , and exaThe total RNA was isolated at different time points after akinete induction  or nitrogen step-down  from wild-type and mutant DR752 cells, using UPzol reagent  according to the manufacturer’s instructions. Briefly, cells were harvested at the different time points, immediately snap-freezed in liquid nitrogen and stored at −80°C till further use. Upon addition of 1 mL UPzol solution, cells were homogenized using glass beads and total RNA was extracted. Concentration and purity of the extracted RNA were estimated using nanophotometer (Implen) and RNA gel-electrophoresis followed by DNase treatment. The genomic DNA contamination was controlled by PCR followed by agarose gel electrophoresis. The DNase-treated RNA was used to generate cDNA using the RT-reaction kit (Applied Biosystems) and 1 μl of this cDNA was used for semi-q RT PCR followed by visualization on an agarose gel. Primers for semi-q RT PCR reactions are listed in Nitrogenase activity was measured with the acetylene reduction method as previously described . Brieflya (Chla) concentration was measured (a was extracted from 1 ml of cultures grown with (BG11) and without nitrogen source (BG110), and from the cultures induced to form akinetes by low light for 2–4 months by adding methanol to a final concentration of 90% (v/v). The suspension was vortexed for 1 min and the cells were centrifuged at 13,000 rpm for 2 min at RT after incubation for 5 min in darkness followed by the measurement of adsorption of the supernatant at 665 nm. The final Chla concentration was calculated using the formula:Thin-layer chromatography (TLC) was performed to analyze the glycolipids composition of heterocysts and akinetes as described  with minmeasured . Brieflya concentration and resuspended in a methanol:chloroform mixture (1:1). Afterward, the lipids in the supernatant were concentrated by evaporation in air under a fume hood. The lipids were dissolved in chloroform and applied on an aluminum plate coated with silica gel . TLC was run in a mobile phase composed of chloroform:methanol:acetic acid:water in a ratio of 23:4:2.7:1. The lipids were visualized by spraying the TLC plate with 25% sulfuric acid and exposing it to 180°C for 60–120 s.For TLC, the cells were pelleted at equal Chla content of akinetes culture was determined as described in previous section. For inducing the akinetes to different stress conditions, 1 ml of akinete cultures with a concentration of 5 μg Chla ml–1 were exposed to either freeze (−20°C) and cold (4°C) conditions or desiccated (centrifuged and pellet dried at 28°C) for 10, 20, and 30 days, respectively. Other stress conditions included the treatment with 10 mM H2O2 for 2 h at 28°C, with 300 μg ml–1 lysozyme overnight at RT in dark and the freeze (in liquid nitrogen for 5–7 min) and thaw (at RT for 20–30 min) cycles for three or six times. For each stress conditions and time points, various dilutions of akinete samples (in the range of 5 μg ml–1−0.5 μg ml–1 Chla) were prepared and 10 μl of each dilution were carefully dropped on agar plates and incubated for 1 week at 28°C under continuous light.The viability of akinetes (3–4 months old) was tested after exposing them to different stress conditions using an agar spot assay. For this, ChlhglB gene Ava_2595 in the genome of A. variabilis. We created the mutant, which will be denoted DR752 from here onward, by insertion of a neomycin resistance cassette (CK.3) into the Ava_2595 gene by double homologous recombination  with the lipids extracted from filaments of the WT and the DR752 strains. In contrast to the WT, the mutant did not show a band with the typical TLC migration property , confirmntiation . As expetep-down . Since tpromoter . OverexpFurthermore, we studied the ultrastructure of the mutant heterocysts by transmission electron microscopy (TEM) of ultrathin sections of mutant and WT filaments . As expeTo assess the oxygen status in the heterocysts of the mutant, the filaments were incubated with triphenyl tetrazolium chloride (TTC), which forms dark red-brown precipitates in a microoxic environment . The WT nifD gene rearrangement required to produce a functional enzyme .Furthermore, we used the acetylene reduction assay to measure the nitrogenase activity under oxic and anoxic conditions . The DR7l enzyme . In contA. variabilis akinete mostly consist of the heterocyst-specific glycolipid HG26-diol  from A. variabilis is the first known gene involved in the envelope formation of akinetes. In this respect, it would be interesting to analyze akinete formation of the previously described hglE mutant of N. punctiforme, which is lacking the HGLs in heterocysts and is affected in diazotrophic growth (In addition to the chemical and structural identity of the laminated layer of both cell types, our study indicates that akinetes and heterocysts use an overlapping biosynthetic pathway to produce the glycolipid envelope, involving the putative polyketide synthase HglB. To our knowledge, c growth .hglB-mutant to unfavorable conditions. The significantly reduced resistance to freezing in the mutant, especially when using multiple freeze-thaw cycles, could be the consequence of a reduced cell wall stability (2O2 could be explained by a thinner physical protective envelope in the mutant akinetes, which makes them more susceptible to damage by reactive oxygen species. The akinetes of the mutant are found to be less stable against any tested stress conditions, except the exposure to cold. Although the mutant develops defective akinetes, they are nevertheless more resistant to extreme environmental influences than vegetative cells due to the presence of the extracellular polysaccharide layer and intracellular adaptations (To illustrate the importance of the glycolipid layers of the akinete cell envelope in stress tolerance and survival, we exposed the akinetes of WT and the tability . Since, tability . This emtability . The redptations . Expecteptations . The thiptations  but the ptations . AltogethglB in akinete and heterocyst envelope formation in A. variabilis. Additionally, we showed that glycolipid layers are necessary to protect the akinetes from various stress factors. The sharing of similar envelope-structures indicates that the developmental processes for akinetes and heterocysts occur in a similar way but fulfill different functions in these two specialized cell types of multicellular cyanobacteria. Further genes known to be involved in heterocyst envelope formation should be studied in the future to confirm the common biosynthetic pathways in heterocyst and akinetes and to learn more about their function in survival of dormant cells. It would also be interesting to understand the upstream divergent signaling pathways and to which point they converge.In summary, this study has established the diverse role of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.RG designed and performed the experiments, interpreted the data, wrote most of the manuscript, drafted the work, made manuscript revisions, and gave final approval. IM designed and supervised the research, wrote part of the manuscript, made substantial contributions to the design of the work, and made interpretation of data for the work. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Certain gastric cancers exhibit some primitive phenotypes, which may indicate a high malignancy. In histologically differentiated early gastric cancer (EGC), the presence and the clinicopathological significance of the primitive phenotype remain unclear.n = 302). For those cases with primitive phenotypes, we analyzed their clinicopathological features and evaluated whether the criteria for endoscopic resection were met.Using immunohistochemical staining we detected the expression of three primitive phenotypic markers SALL4, Glypican-3(GPC3), and AFP in whole tissue sections of differentiated EGC . Multivariate analysis revealed that presence of primitive phenotypes was an independent risk factor for LNM . Interestingly, we found 2 cases with primitive phenotypes developed LNM, and they both met the expanded indications of endoscopic resection for differentiated EGC.We found that 9.3% (28/302) of all differentiated EGC cases have primitive phenotypes, and most of these cases (25/28) exhibit a histomorphology similar to conventional differentiated EGC. Patients with primitive phenotypes had a deeper invasion, a higher rate of ulcer and lymphatic invasion than cases without primitive phenotype. Moreover, patients with primitive phenotypes displayed a significantly higher frequency of LNM than those without (57.1% vs 8.8%, A small number of differentiated EGC have primitive phenotypes, which were closely related to LNM and were an independent risk factor for LNM. Given its highly aggressive behavior, differentiated EGC with primitive phenotypes should be evaluated with stricter criteria before endoscopic resection, or considered to give an additional surgical operation after endoscopic resection. Early gastric cancer (EGC) refers to gastric cancer that does not invade deeper than the submucosa, regardless of lymph node metastasis (LNM). Nowadays, endoscopic resection, including endoscopic mucosal resection and endoscopic submucosal dissection, has become the standard treatment for EGC . The effEndoscopic resection of EGC is applied based on the premise that the patients do not have LNM, and it is vital to assess the risks of LNM for EGC. At present, indications for endoscopic resection have been established, and these indications can evaluate LNM risks of EGC. Several clinicopathological variables, such as tumor size, depth of invasion, ulcer presence, lymphatic/vascular invasion and tumor differentiation are included in these indications. As to tumor differentiation, gastric cancer can be classified as the differentiated or undifferentiated based on its histologic appearance: if cancer cells form a tubular or papillary structure, it is classified as differentiated gastric cancer; if gastric cancer consists of signet ring cell carcinoma or mucinous adenocarcinoma, it is classified as undifferentiated gastric cancer . Most ofRecently, accumulating evidence indicated that certain gastric adenocarcinomas have some primitive phenotypes, and this subtype of gastric cancer displays a high malignancy , 7. A stFor gastric cancer with primitive phenotypes, histomorphology may not reflect the true degree of differentiation, and GAED is a representative example: it is histologically similar to conventional differentiated gastric adenocarcinoma with a tubular or papillary structure, and GAED ought to be classified as differentiated gastric cancer according to its tubular or papillary histology. However, GAED displays the phenotypes of immature differentiation, and primitive phenotypic markers  are frequently expressed in GAED –14. The This study included 302 cases of differentiated EGC that underwent surgical resection from 2008 to 2018 in our hospital. All specimens and patient information were obtained with institutional review board approval. Postoperative pathological examinations confirmed that all cases were differentiated EGC. Averagely, 16.9 lymph nodes were retrieved from each case (range: 2–38).EGC is defined that cancer cells invade not deeper than the submucosal layer. T1a gastric cancer means tumor invades the lamina propria and or muscularis mucosae, while T1b gastric cancer means tumor invades into the submucosal layer. For T1b, if the tumor invasion frontier is less than 500 μm from the mucosal muscle layer, it is classified as SM1, and if it is greater than or equal to 500 μm, it is classified as SM2. The identification of differentiated gastric cancer is based on criteria previously published [The archived paraffin blocks from all 302 cases were re-sectioned with a thickness of 4 μm, and immunohistochemical staining was performed on the whole tissue sections. The sections were deparaffinized and hydrated, and then antigen retrieval was performed . After quenching endogenous peroxidase activity, sections were incubated with primary antibodies for 12 h at 4 °C. Thereafter, sections were incubated with an anti-mouse polymer kit  for 30 min at room temperature. Then slides were visualized with DAB and counterstained with hematoxylin. Primary antibodies included: mouse anti-human SALL4 antibody , mouse anti-human GPC3 antibody , mouse anti-human AFP antibody , and mouse anti-human D2–40 antibody .Appropriate positive and negative controls were included for each run of immunohistochemical staining, and incubation of samples with antibody dilution buffer without the primary antibodies, was employed as the negative staining control. The positive staining control for the SALL4 antibody, were carcinoma tissues from gastric hepatoid adenocarcinoma, which had been demonstrated strongly positive for SALL4. The positive control for AFP and GPC3 antibodies, were 2 cases of poorly differentiated hepatocellular carcinoma with strong expression of AFP and GPC3.The stained sections were evaluated by two senior pathologists. When there was a disagreement, they review the sections together and discussed to reach an agreement. A positive result was confirmed if SALL4 expression was localized in the nucleus, AFP was localized in the cytoplasm, and GPC3 and D2–40 were localized in the cell membrane and cytoplasm. When measuring the expression intensity of the markers SALL4, GPC3, and AFP, a 3-point scoring system was used : negativFor PAS staining, sections of archived paraffin block (thickness 4 μm) were deparaffinized and placed in water. A solution of 1% periodic acid was added for 10 min, and then sections were incubated with Schiff solution for 15 min for staining. For D-PAS staining, sections were incubated with amylase at 37 °C for 15 min, and then PAS staining was performed as described above.According to previous literature , 16, indP < 0.05 was considered statistically significant.Categorical variables were compared using chi-square or Fisher exact test, and agreement between two variables was evaluated with Kappa consistency test. Univariate and multivariate logistic regression were used to analyze the risk factors associated with LNM. The statistical software was SPSS 22.0, and Immunohistochemistry in whole tissue sections reveal that among 302 cases of differentiated EGC: 1) 24 cases were SALL4 positive, of which 13 were diffusely positive and 11 were focally positive; 2) 8 and 2 were positive for GPC3 and AFP respectively, and both GPC3 and AFP were focally positive , and there also existed an association between AFP expression and GPC3 expression . Of the 8 patients who were GPC3-positive, 3 were SALL4-positive , and GPC3 expression was related to SALL4-diffuse expression .According to Table Morphologically, all the 28 cases with primitive phenotypes displayed a tubular, cribriform, or papillary morphology, and the nuclei of the cancer cells were moderately atypical , which was significantly higher than that of those without primitive phenotypes . As LNM were present in 40 cases of all the 302 patients, we can deduce that 40% (16/40) of differentiated EGCs with LNM have primitive phenotypes.P = 0.530), and LNM rates were higher in these two groups than in T1a. Therefore, they were combined into one group (T1b group). In terms of tumor site, we found that tumors located in the upper stomach had significantly lower LNM rates than tumors located in the middle and lower stomachs. There was no significant difference in the rates of LNM in cases located in the middle and lower stomachs (P = 0.554), so these cases were also combined into one group. We also found that tumor size and lymphatic involvement were also closely related to LNM. Table In terms of invasion depth, there was no significant difference in LNM rates between SM1  and SM2  P = 0.50, and LNP = 0.001, HR 6.977, 95% CI: 2.199–22.138), tumor located in the middle and lower stomach , and LVI  were independent high risk factors for LNM. Therefore, presence of the primitive phenotype was a useful marker to predict LNM risks for differentiated EGC.According to the univariate analysis, five factors  were closely related to LNM, these factors were further included for the multivariate analysis. As a result, we found that presence of the primitive phenotype (P = 0.002). Moreover, when the expanded indications was met, the cases with primitive phenotypes were more prone to develop LNM than those without . Table Next, we investigated whether the 302 cases met the indications of endoscopic resection and the occurrence of LNM. Of the 28 cases with primitive phenotypes, we found that: 22 cases  did not meet the endoscopic resection criteria (absolute and expanded indications); 2 cases met the absolute indications and had no LNM; 4 cases met the expanded indications for differentiated EGC (Ex-SM1). Interestingly, 2 of the above mentioned 4 cases had LNM. Of the 274 cases without primitive phenotypes, 132 cases  did not meet the indications for endoscopic resection; 57 met the absolute indications and none of them had LNM; 85 met the expanded indicationsfor differentiated EGC and 1 of them had LNM. As a result, the proportion of patients who did not meet the indications for endoscopic resection was significantly higher in patients with primitive phenotypes than in those without  of histologically differentiated EGC, and cases with primitive phenotypes had a significantly higher frequency of LNM than those without (57.1% vs. 8.8%). Furthermore, our results revealed that presence of the primitive phenotype was an independent high risk factor for LNM in differentiated EGC.As the presence of primitive phenotype was closely related to LNM, it is necessary to determine the primitive phenotype in differentiated EGC. We found that most of the differentiated EGC with primitive phenotypes lacked recognizable histological features, and only 3 of the 28 cases with primitive phenotypes had characteristic morphology (cancer cells that are clear or vacuole-like) consistent with GAED, while the remaining 25 cases were morphologically difficult to distinguish from conventional differentiated EGC. Therefore, histologic morphology cannot indicate the presence of primitive phenotypes, and it should be determined through immunohistochemical assay of SALL4, GPC3, and AFP. Meanwhile, we found the expression of SALL4 and GPC3 are closely related to that of AFP, and all the AFP-positive cases were also SALL4-positive, which is consistent with previous studies , 11. As As shown in Table At present, the indications of endoscopic resection for EGC proposed by the Japanese Gastric Cancer Association are crucial for screening patients suitable for endoscopic resection. Many studies have proven that the indications can minimize the risk of LNM. LNM rates are extremely low in those who meet absolute endoscopic resection criteria , 17, andIn our cohort, differentiated EGC with the primitive phenotypes was significantly associated with a deeper invasion and an increased rate of LNM, indicating its highly aggressive behavior and high- grade malignancy. Although differentiated EGC with primitive phenotypes exhibit a well differentiated or moderately differentiated histological morphology, it seems reasonable to classify these cases as undifferentiated adenocarcinoma considering its high malignancy, aggressive behavior, as well as the expression of the primitive markers. If differentiated EGC with the primitive phenotypes were classified as undifferentiated gastric cancer, and assessed with the expanded indications for undifferentiated EGC , then the abovementioned 2 cases with LNM would not meet Ex-3, and the clinical risk that omitting LNM undiscovered and untreated after endoscopic resection would be avoided.In summary, we found a few differentiated EGC have primitive phenotypes, which were closely related to LNM. Given its highly aggressive behavior, differentiated EGC with primitive phenotypes should be evaluated with stricter criteria before endoscopic resection, or considered to give an additional surgical operation after endoscopic resection.


Background: Subarachnoid hemorrhage (SAH) caused by rupture of an intracranial aneurysm, is a life-threatening emergency that is associated with substantial morbidity and mortality. Emerging evidence suggests involvement of the innate immune response in secondary brain injury, and a potential role of neutrophil extracellular traps (NETs) for SAH-associated neuroinflammation. In this study, we investigated the spatiotemporal patterns of NETs in SAH and the potential role of the RNase A  application on NET burden.Methods: A total number of n=81 male C57Bl/6 mice were operated utilizing a filament perforation model to induce SAH, and Sham operation was performed for the corresponding control groups. To confirm the bleeding and exclude stroke and intracerebral hemorrhage, the animals received MRI after 24h. Mice were treated with intravenous injection of RNase A (42μg/kg body weight) or saline solution for the control groups, respectively. Quadruple-immunofluorescence (IF) staining for cell nuclei (DAPI), F-actin , citrullinated H3, and neurons (NeuN) was analyzed by confocal imaging and used to quantify NET abundance in the subarachnoid space (SAS) and brain parenchyma. To quantify NETs in human SAH patients, cerebrospinal spinal fluid (CSF) and blood samples from day 1, 2, 7, and 14 after bleeding onset were analyzed for double-stranded DNA (dsDNA) via Sytox Green.Results: Neutrophil extracellular traps are released upon subarachnoid hemorrhage in the SAS on the ipsilateral bleeding site 24h after ictus. Over time, NETs showed progressive increase in the parenchyma on both ipsi- and contralateral site, peaking on day 14 in periventricular localization. In CSF and blood samples of patients with aneurysmal SAH, NETs also increased gradually over time with a peak on day 7. RNase application significantly reduced NET accumulation in basal, cortical, and periventricular areas.Conclusion: Neutrophil extracellular trap formation following SAH originates in the ipsilateral SAS of the bleeding site and spreads gradually over time to basal, cortical, and periventricular areas in the parenchyma within 14days. Intravenous RNase application abrogates NET burden significantly in the brain parenchyma, underpinning a potential role in modulation of the innate immune activation after SAH. Subarachnoid hemorrhage (SAH) caused by rupture of an intracranial aneurysm, is a life-threatening emergency that is associated with substantial morbidity and mortality . ApproxiAmong these immune reactions, recent studies suggest that neutrophils play a particular role in SAH induced inflammation . Neutropin vivo SAH models  signals such as extracellular RNA (exRNA) have been described as a key player in the involvement of central system pathologies, including hemorrhagic stroke . In thisH models . Moreovein vivo model of SAH. We report that NET accumulation begins in the ipsilateral side of bleeding, and spreads over time to the parenchyma on both hemispheres, peaking on day 14 after SAH. To confirm these findings in the human system, we also measured NET burden in cerebrospinal spinal fluid (CSF) and blood samples in SAH patients and show that NET accumulation occurs in both compartments significantly. Furthermore, we report that RNase significantly reduces NET formation in the parenchyma, thus being an attractive mediator for evaluation in subsequent studies.In this study, with regard to the migratory characteristics of neutrophils, we investigated the spatial and temporal pattern of NET formation in an The analysis on human samples was approved by the local ethics committee of Charité University Hospital . All patients or their authorized individuals gave written consent to the collection of blood and CSF samples. All animal experiments were approved by the Regional Office for Health and Social Affairs  and were performed in conformity with the German law of animal protection and the National Institute of Health Guidelines for care and use of laboratory animals.Peripheral blood and CSF samples were collected from patients with aneurysmal SAH on day 1, 2, 7, and 14 after SAH onset. For each time point, 2ml peripheral blood was drawn using Ethylene Diaminetetraacetic acid (EDTA) tubes, and 2ml CSF was collected from the initial placement of an extraventricular drain (EVD) at onset of SAH, and then further collected from the existing EVD. Some patients also had additional lumbar drainage, but in this study only ventricular CSF was used. Blood and CSF samples were immediately placed on ice and spun down twice at 4°C at 500g for 5min, and supernatant was frozen at −80°C before further analysis. Double-stranded DNA (dsDNA) was quantified in CSF and plasma samples as previously published . In detan=81 mice were included in this study.Male C57BL/6-mice were kept at the animal facility at the Neuroscience Research Center at Charité University . Their age ranged from 8 to 12weeks and with a weight range of 18–28g. Animals underwent SAH operation, and sham operation (as the control condition), and were sacrificed at three different time points  after operation. A total number of Subarachnoid hemorrhage was induced with a filament perforation model as described previously . Briefly2/N2O+isoflurane gas anesthesia. The animals’ respiration was observed with Small Animal Monitoring System , while their temperature was maintained via controlled warming blankets. The presence of SAH was validated from T2 weighted image. Here, SAH volume was estimated based on the formula n corresponds to the bleeding area on each coronal MRI and d corresponds to the MRI slide thickness.All SAH mice received an MRI 24h after surgery to confirm the bleeding and animals who had a stroke or intracranial hemorrhage (ICH) were excluded from the experiments . The 1H via the sublingual vein. The further injections were repeated every 3days in the tail vein until scarification as previously reported  received treatment with RNase A (Thermoscientific EN0531), which was administered intravenously (42μg/kg body weight) and intravenous application of saline solution was used as a control, as described previously . The firreported . The totTo preserve the SAS, whole skulls were harvested and kept in an ascending concentration series of sucrose for proper dehydration  at 4°C and then snap frozen with isopentane. The skulls were then embedded in Tissue-Tek O.C.T. Compound  and carefully cut into 10μm thin slices with Microm Sec35 blades (Termo APD Consumables) using a cryostat (Thermo Fisher Scientific Inc. Microm HM 560).Brain sections were blocked for 30min with 5% bovine serum albumin (BSA) in Tris-Buffered Saline with 0.05% Tween (TBST) at room temperature. After that, primary antibodies against citrullinated H3 , NeuN , and F-actin  diluted in 5% BSA/TBST were added for incubation at 4°C over night. The next day, sections were washed three times in 5% BSA/TBST for 10min, followed by 1.5 h of incubation with the secondary antibodies Rhodamine Red-X-conjugated Donkey IgG anti-Mouse  and Alexa Fluor 647-conjugated Donkey IgG anti-Rabbit IgG , diluted in 5% BSA/TBST. After secondary antibody incubation, specimens were washed three times in PBS for 5min and mounted with DAPI Mounting Medium . The sections were then imaged with confocal microscopy (Leica DM 6000/SP5) and analyzed with ImageJ. Amount of NETs was determined by calculating the ratio area covered by citH3 over total area covered by phalloidin, NeuN, and DAPI .t-tests for the comparison of two groups. The values are displayed as means±SEs and values of p<0.05 were considered statistically significant. Elements of BioRender.com.Data were analyzed using GraphPad Prism for statistical analyses . ANOVA analyses were used to compare multiple, unpaired in vivo, we utilized a filament perforation model to induce subarachnoid hemorrhage in C57BL/6 mice, while Sham operation was performed as a control condition. MRI was conducted 24h after bleeding to verify SAH and exclude other pathologies such as stroke or intracerebral hemorrhage (via immunofluorescence staining using citrullinated Histone (citH3), a known specific marker for NET remnants , while in the parenchyma, NET levels increase gradually over time in basal, cortical, and periventricular compartments distant to the region of bleeding. These findings are supported by a recent study reporting NET accumulation after SAH as well as their involvement in neuroinflammatory events, albeit the time point of the peak of NETs differed to some degree . Interesfter SAH .via DNase has been postulated as a potential mechanistic treatment strategy to reduce NET burden . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Landesamt für Gesundheit und Soziales (LaGeSo) Berlin (Germany).KT, FS, AF, SL, and RX conducted the experiments and analyzed the data. RX, AF, and PV designed the study. All authors contributed to the article and approved the submitted version.RX was supported by the BIH-Charité Clinician Scientist Program funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health. We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charité – Universitätsmedizin Berlin. KT is supported by the Berlin Institute of Health (BIH) Research Stipend as well as the Sonnenfeld Foundation.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.


In this paper, a lead–bronze/steel bimetal composite was produced by vacuum diffusion welding technology. The microstructure, hardness and tribological properties under the dry sliding condition of the bimetal structured material were investigated and compared with two reference samples, i.e., lead–bronze and Mn/Si–brass. The wear mechanism of the three materials was also analyzed in detail. It was found that the bimetallic structure possessed the best wear resistance among the three samples. When paired with the ball bearing steel, the wear rates of the lead–bronze and Mn/Si–brass were 13 and 54 times higher than that of the bimetal composite. When paired with bearing steel, the wear rates of the two materials were 13 and 54 times higher than the bimetallic composite, respectively. This is because the steel layer served as a bearing layer to decrease the plastic deformation of the bronze layer. Furthermore, the lead can accelerate the formation of a dense hardened layer at the sliding interfaces to avoid subsequent wear of the bronze surface. Nevertheless, this hardened layer caused severe scuffing on the steel balls. Therefore, lead–bronze/steel structured material is recommended to match with hard counterface material, such as cemented carbide. Axial piston pumps are widely used in high-duty hydraulic fluid power systems due to their advantages of high-power densities, high-limit load pressure, and high overall efficiency ,2,3. OneHard coatings such as TiN , CrSiN , CrZrN , and DLC5Si3 precipitated particles [Copper alloys are applied in axial piston pumps due to their high thermal conductivity, mechanical strength, good machinability, etc. ,12. Amonarticles . Other tarticles ,21,22, farticles ,24,25, darticles ,27,28,29articles  are low-Among these technologies, vacuum diffusion welding has various advantages: low heating temperature, little effect on the properties of the substrate material, similarity in performance of the welded joint with respect to the substrate material, and small deformation . HoweverIn this paper, a lead–bronze layer with a millimeter-scale was jointed with a steel backboard by vacuum diffusion welding technology. This low-lead copper-based material acted as friction pairs of axial piston pumps operated under extreme working conditions. The tribological properties of the lead–bronze/steel composite were investigated and compared with those of two reference materials, i.e., lead–bronze and Mn/Si-modified brass. The wear mechanisms of these materials were also discussed and analyzed. ®) and lead-free brass with Mn and Si (ZY331604®) plates were obtained from Ningbo Zycalloy Co., Ltd., Ningbo, China. The chemical composition of these copper alloys is listed in Commercial lead–bronze  and 42CrMo steel plates (8 mm in thickness) were abraded using 400# sandpaper to obtain a surface roughness (Ra) of c.a. 0.6 μm. Both plates were immersed in 10% NaOH solution at 80 °C for 10 min to remove the residual grease on the surface. Next, they were immersed in 10% HCl aqueous solution for about 10 min to remove the outer oxide layer. Finally, all metal plates were rinsed with deionized water and alcohol, respectively, and then dried for future use. A vacuum diffusion furnace  was used to prepare bimetallic alloy by vacuum welding technology. During this vacuum welding process, two types of metal plates were put into the furnace with the bronze plate on the bottom and the steel plate on the top. A pressure of 6 MPa was loaded on the two plates, as shown in 3 N−1 m−1); V is the wear loss volume (μm3); F is the normal load (N); and S is the sliding distance (m).The microstructure of the specimens was observed by an optical microscope  and a scanning electron microscope (SEM) with Energy Dispersive X-ray Spectroscopy (EDX). An EM-30AX Table-Top scanning electron microscopy (SEM) from COXEM Co. Ltd., Daejeon, Korea, equipped with an Oxford Xplore Compact 30 energy dispersive X-ray (EDX) spectrometer was operated at an electron accelerating voltage of 20 KV in secondary electron (SE) scanning mode. The work distance was 9 mm for EDX detecting. Before SEM analysis, the samples were mounted on an aluminum stub with carbon adhesive tape. Before the microstructure characterization, the top surface of samples was etched by acid solution. Before the examination of cross-sections of the bimetallic specimen, the cross-sectional samples were mounted with resin, well-polished and etched by acid solution. A Vickers microhardness tester was used to measure the hardness of the specimens under 0.098 N load with 12 s holding time. Ten points were measured on each sample to obtain the average hardness. The tribological properties of the specimens were examined by a pin-on-disc tribometer. The tests were conducted under the dry sliding test condition, with a sliding speed of 1000 revolutions/min (0.314 m/s). Two types of counterface balls with 6 mm in diameter were used in the tribological tests, of which one was made from ball bearing steel (GCr15 steel: C 1.0% and Cr 1.5% with HRC = 62) and another was cemented carbide (WC 94% and Co 6% with HRC = 92). The normal load was 1 N, i.e., 500 MPa contact pressure calculated by the Hertzian contact theory. The sliding tests were conducted for 20,000 revolutions (377 m). The wear tracks and worn counterface balls were investigated by a confocal microscopy  and SEM with EDX. The wear rate was calculated by the following equation:5Si3 hard particles were evenly distributed into the matrix of α Phase and β phase [ β phase . The graThe average hardness results for three samples were demonstrated in The coefficient of friction (COF) curves of the three samples are shown in To reveal the wear mechanism of these materials, SEM micrographs of the corresponding wear tracks are shown in 5Si3 accumulate at the sliding interface. This can also explain the gradually increasing behavior of the COFs for this sample, as shown in For the Mn/Si–brass sample b,e, littThe surface of the bimetallic sample was only polished slightly with a smooth and dense layer observed on the wear tracks c,f. The Next, the tribological properties of three samples against the WC-based balls, which had higher hardness and more inert chemical properties, were investigated. The COF curves for these samples against WC balls under 1 N are given in (1)The lead–bronze/steel bimetallic structure was produced by a vacuum diffusion welding technology. The lead–bronze layer was tightly bonded with steel and formed a continuous diffusion layer.(2)The tribological properties of three samples, i.e., lead–bronze, Mn/Si brass and bimetallic materials were tested. Under a dry sliding condition and high load (~500 MPa), large amounts of plastic deformation occurred for the lead–bronze, which was due to its insufficient mechanical strength and the failure to form a lubricating film by the soft lead phase. The wear debris with lead was easy to be hardened and accelerated the adhesive wear and abrasive wear. This situation was more serious when the counterface material was a relatively soft metal (GCr15).(3)Although the lead-free Mn/Si–brass alloy had higher hardness, its wear performance was worse than the lead–bronze under the dry sliding condition, which was due to the severe abrasive wear derived from hard precipitations in the alloy.(4)The lead–bronze/steel bimetallic structure can minimize the use of lead in the copper alloy. Meanwhile, this bimetallic structure achieved a better wear resistance than the other two alloys. The steel layer can serve as a bearing layer to decrease the plastic deformation of the bronze layer and accelerate the formation of a dense hardened layer at the interfaces to avoid subsequent wear of the bronze surface. Nevertheless, this hardened layer caused severe scuffing on the counterface materials.(5)Therefore, lead–bronze/steel-structured material had the best wear resistance among the three materials. It is recommended to be applied in the key friction pairs of Axial piston pumps matched with hard counterface material, such as cemented carbide.


Using an experimental video recording trial, feed conversion ratio (FCR), body weight gain (BWG), residual feed intake (RFI) and feed intake (FI) traits were recorded in 40 full-sibling families from the GIFT  Nile tilapia breeding population. Fish were genotyped with a ThermoFisher Axiom 65 K Nile tilapia SNP array. Significant heritabilities, ranging from 0.12 to 0.22, were estimated for all the assessed traits using the genomic relationship matrix. A negative but favourable genetic correlation was found between BWG and the feed-efficiency related traits; −0.60 and −0.63 for FCR and RFI, respectively. While the genome-wide association analyses suggested a polygenic genetic architecture for all the measured traits, there were significant QTL identified for BWG and FI on chromosomes seven and five respectively. Candidate genes previously found to be associated with feed-efficiency traits were located in these QTL regions, including ntrk3a, ghrh and eif4e3. The accuracy of breeding value prediction using the genomic data was up to 34% higher than using pedigree records. A SNP density of approximately 5,000 SNPs was sufficient to achieve similar prediction accuracy as the full genotype data set. Our results highlight the potential of genomic selection to improve feed efficiency traits in Nile tilapia breeding programmes.Nile tilapia is a key aquaculture species with one of the highest production volumes globally. Genetic improvement of feed efficiency via selective breeding is an important goal, and genomic selection may expedite this process. The aims of this study were to  One of the key features of aquaculture species compared with terrestrial farmed species is their greater feed efficiency (FE) . For exaOreochromis niloticus) is the third most produced farmed species globally (1) dissect the genetic architecture of feed-efficiency traits in a Nile tilapia breeding population, 2) map the genomic regions associated with these traits and identify candidate genes, 3) evaluate the accuracy of prediction of breeding values using genomic data, and 4) assess the impact of the SNP density on the genomic prediction accuracies. To the best of our knowledge, this is the first study to assess genomic prediction for feed-efficiency traits in Nile tilapia. Our results highlight the feasibility of including feed-efficiency traits into Nile tilapia breeding programs. Furthermore, they show the potential improvement in breeding value prediction using low density SNP panels compared to using pedigree methods, which can enhance genetic gain and improve production efficiency.Nile tilapia  breeding program based in Jitra, Malaysia, and managed by WorldFish (This study used tissue samples archived by orldFish  These fi1) Generally 1 week is sufficient for an adaptation period. However, this was increased to 2 weeks as a precaution because some fish could adapt faster than others. The 10 days of fasting 2) represents the longest period permitted while maintaining acceptable fish welfare, while fewer days would probably have a low impact in terms of weight loss. The longest period was ascribed to the feeding stage 3), which allows the fish to recover from the fasting period but also enables successful measurement of compensatory growth, ensuring that this does not impact on the measurements of the feeding and growth during the last stage. Previous results by Full details of the experimental challenge are given in previous studies, which reported methodology development and quantitative genetic analysis of feed efficiency traits ; 2018b. Body weight was measured at the beginning and end of each stage. Due to aggression between fish, only 1,029 fish were available at the beginning of the FI stage where fish were fed twice a day (07:00 and 13:00), pellet by pellet, and videos of each meal were recorded. Fish received a total of 13 meals during this stage . Uneaten pellets were removed at the end of each meal. The number of pellets consumed by each fish was counted through video analysis. Throughout the experiment, water temperature was fixed at 28°C ± 1°C, while the photoperiod was 12L:12D cycle. The phenotypic sex of each fish was registered by visual observation of the gonads at the end of the FI recording period. The fish were sufficiently developed to show sexual differentiation, but were not yet exhibiting any reproductive behaviour. Mortality was recorded daily throughout the challenge. Thirty two fish died during the FI stage and were not included in the analyses.As a practical calculation of pellet weight, a total of 500 pellets (16.4 ± 1.76 mg) were weighed, and as the variation in weight per pellet was considered low enough, the average weight of each pellet was used. Thus, the FI in grams for each fish could then be calculated as the sum of pellets consumed during the 13 meals. Body weight gain (BWG) was calculated during the FI stage as the difference between the body weight at the end and at the beginning of this stage. The quotient between these two traits (FI/BWG) was used to estimate the feed conversion ratio (FCR). Finally, residual feed intake (RFI) was estimated as the difference between the amount of feed intake by each fish and the amount predicted .Outliers were highlighted using the boxplot. stats function of the R package “stats”  and were® SNP array (1) call rate <0.95, 2) minor allele frequency (MAF) < 0.05 or 3) deviation from Hardy-Weinberg Equilibrium (p < 1 × 10−6). Simultaneously, animals with a call rate <0.95 were also excluded. Finally, to excluded potentially duplicated samples, both of each pair of fish with a proportion of IBD higher than 0.7 were removed from further analyses. Thus, a total of 755 fish and 48,431 SNPs remained after these filters, representing the final data for the SNP array  by using a 65 K AxiomNP array . The rawNP array  was perfNP array ..To assess the genetic structure within the Nile tilapia population, a principal component analysis (PCA) was performed through PLINK v1.09. The two main components were plotted along the two axes in R.c is the vector of random effect associated with the common environmental effects (aquarium effect), e is the vector of random residuals errors, whereas ,W are the incidences matrices for the fixed, genetic and environmental effects, respectively. A normal distribution was assumed for the random genetic, common, and residuals effects, with a mean of zero and the following variance:I are the numerator relationship and identity matrix, respectively. For all the traits, heritability was estimated as follows:Only fish with phenotype and genotype data were used for the pedigree-based BLUP (PBLUP) model, to enable a fair comparison between pedigree and genomic prediction performance. To predict the Estimated Breeding Values (EBVs) and estimate the variance components, the following univariate linear model was used:(G) relationship matrix. In this case, the vector of random additive genetic polygenic effects g has a distribution ∼ N  model was the same as for PBLUP and GBLUP. However, a combined kinship matrix H  which in program .1)Random animal: for each replicate, 80% of all the fish were randomly drawn independently of their family, and used as a training dataset. The remaining 20% of the samples were used as a validation dataset.2)Random families: for each replicate, 80% of the families were randomly drawn and used as a training set. For the remaining 20% of the families, the phenotype was masked and used as a validation dataset. Thus, breeding value predictions were made in the validation set using phenotypic data from different families.The ability to predict the breeding values for the traits using pedigree and genomic approaches was assessed using a fivefold cross-validation approach. To assess the predictive ability among the different models, a total of 100 replicates were used. For each replicate, 80% of fish were used as a training dataset and, for the remaining 20%, the phenotypes were masked and used as the validation dataset. For each model, two cross-validation approaches were applied:For each approach, prediction accuracy was calculated as follows:p values were adjusted by the number of independent tests.Significant differences in predicted accuracies among models were assessed for each trait using both approaches. A Shapiro-Wilk test was done to evaluate the normality of the distribution of the predicted accuracies. The “rstatix” R package , was useSince the relatively high cost of high density SNP array genotyping may present a barrier to routine genotyping of large number of animals within a population, the efficacy of reduced density SNP panels for predicting breeding values was assessed. A total of 23 subsets of random SNPs were selected from the high density SNP array. These densities included 0.1, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 and 90% of the entire SNP dataset. For each SNP density, 100 replicates of a random animal five-fold cross validation were generated. The GBLUP model fitted for each density is the same as that described for the full genotype data set, and the accuracy was predicted as detailed previously.leaving-one-chromosome-out (LOCO) approach from the GCTA software v.1.92.2  and used as the basis for a literature search for relevant functions connected to feed intake and efficiency. This size was selected based on Summary statistics for the feed efficiency and growth traits calculated after the trial are shown in The additive genetic variance estimated through PBLUP models was higher compared with GBLUP and ssGBLUP. The PBLUP model also had the lowest estimates for the environmental and residual variance .2 among models.Heritability estimates using PBLUP, GBLUP and ssGBLUP were significant, and low to moderate in magnitude . In all A high genetic correlation of 0.98 ± 0.03 between FCR and RFI was found, suggesting that both measurements are essentially representations of the same trait . NegativOni5 associated with both FCR and RFI  and growth hormone-releasing hormone (ghrh) were found within the QTL for BWG, eif4e3 being situated approximately 89 Kbp upstream from the most significant SNP (AX-317169164), whereas ghrh is ∼2 Kb upstream of the SNP AX-317150877. In the case of FI, the neurotrophic tyrosine kinase receptor type 3 (ntrk3), hyaluronidase 4  and Sus scrofa sperm adhesion molecule 1 (spam1), genes were found within the QTL region.The genes within a 500 Kb window surrounding the most significant SNPs associated with these traits are summarized in Two different cross-validation approaches were tested to estimate prediction accuracies; a random individual and a random family approach. For the former, higher accuracies were achieved for BWG and FI when genomic data were used, with an increase of 34% compared with PBLUP . HoweverAs expected, all the models resulted in lower prediction accuracies when a random family approach was used, compared with the random animal approach. Furthermore, two main trends were observed. Firstly, for FCR and BWG, there were no significant differences in prediction accuracies using any of the models incorporating genomic data . SecondlA decrease in the estimated heritabilities were observed with the low density panels (data not shown). Therefore, for the calculation of prediction accuracy at all SNP densities, a single heritability estimated with all the available markers was used, as is considered the most accurate estimate . Only a Tilapia  population (Oncorhynchus mykiss), but not for FI, whereas Genetic parameters estimates for FE traits are relatively rare in aquaculture species, but have been extensively assessed in livestock species with significant heritabilites detected, ranging from 0.13 up to 0.84 . The stupulation . FurtherDicentrarchus labrax) and rainbow trout  would result in favourable correlated responses for feed efficiency traits within the current population. Although these results are in agreement with previous studies in sea bass , several QTLs were detected for ADG, feed conversion efficiency and feed intake . Fish and higher vertebrates growth is partly controlled by the GH/insulin-like growth factor-I (IGF-I) axis, which is also involved in the regulation of several physiological process in fish such as lipid and protein metabolism, immune function and feeding behaviour , a hybrid fish with an increased growth rate compared with their parental species.Another interesting candidate gene, response . PreviouNeurotrophic Tyrosine Kinase Receptor Type 3, ntrk3, the ligand of neurotrophin 3 (ntf3) may act as a modulator of feeding and satiety in mice and is associated with eating disorders in humans  are likely to be explained by the genetic distance between fish from the training and validation groups. It has been shown that predicting EBV using data from fish from different full-sib families (i.e. random-family approach) affects mainly the pedigree-based model and traits that are not possible to measure on the candidate themselves . HoweverThe impact of SNP density on predicted accuracies has been studied thoroughly for a wide variety of traits and species in aquaculture . The resSignificant heritabilities were confirmed for feed-efficiency traits in a Nile tilapia breeding population, highlighting that genetic improvement is feasible. A negative, but favourable, genetic correlation was detected between BWG and FCR using the genomic data, suggesting selection for BWG may indirectly improve FCR. The traits were polygenic, suggesting the genomic selection may be the most effective route to incorporating genotype data into selection decisions. Genomic prediction markedly outperformed pedigree-based prediction, and this was the case even for relatively low density SNP markers. This study therefore highlights the potential for genomic selection to improve feed efficiency traits in Nile tilapia breeding programmes.