N-acetyl-para-amino phenol , which is commonly used for its analgesic and antipyretic properties may lead to hepatotoxicity and acute liver damage in case of overdoses. Released cytokines and oxidative stress following acute liver damage may affect other organs’ function notably the diaphragm, which is particularly sensitive to oxidative stress and circulating cytokines. We addressed this issue in a mouse model of acute liver injury induced by administration of APAP. C57BL/6J mice (each n = 8) were treated with N-acetyl-para-amino phenol (APAP) to induce acute drug caused liver injury and sacrificed 12 or 24 h afterwards. An untreated group served as controls. Key markers of inflammation, proteolysis, autophagy and oxidative stress were measured in diaphragm samples. In APAP treated animals, liver damage was proven by the enhanced serum levels of alanine aminotransferase and aspartate aminotransferase. In the diaphragm, besides a significant increase in IL 6 and lipid peroxidation, no changes were observed in key markers of the proteolytic, and autophagy signaling pathways, other inflammatory markers and fiber dimensions. The first 24 h of acute liver damage did not impair diaphragm atrophic pathways although it slightly enhanced IL-6 and lipid peroxidation. Whether longer exposure might affect the diaphragm needs to be addressed in future experiments. APAP is highly used due to its analgesic and antipyretic properties and while safe and effective at therapeutic doses, its overdose may lead to hepatotoxicity and acute liver damage. At therapeutic doses, most of APAP is metabolized by phase II conjugating enzymes and excreted as a non-toxic compound. The remaining APAP is degraded via the cytochrome p-450 system into a more toxic substance, N-acetyl-p-benzoquinone imine, whose toxic action can be prevented by glutathione2. In APAP overdose, glutathione scavenger reserves fade and, once depleted, the toxic metabolite damages liver cells, especially mitochondria where it leads to mitochondrial membrane permeabilization and dysfunction3.Drug-induced liver injury represents a serious problem growing in prevalence and importance. Acute liver damage is induced by several toxic agents, of which N-acetyl-para-amino phenol  is the most common drug responsible for toxic liver injury5. In acute damage, the liver releases cytokines into the circulation5 and secondarily may influence other organs’ function and biology.Hence, acute liver injury can end as a systemic disease with decreasing liver synthesis and detoxification function and increasing cytokine production. Several authors pointed to the fact that acute liver injury is probably not an entity that remains limited to the liver7 and diaphragm weakness with muscle atrophy through activation of several proteolytic pathways and autophagy has been reported in situations associated with enhanced circulating cytokines and/or oxidative stress9.The diaphragm is particularly sensitive to oxidative stress and circulating cytokinesWe therefore hypothesized that acute liver injury may affect the diaphragm and induce atrophic signaling via protease-based proteolysis and autophagy. This issue was addressed in a mouse model of acute liver injury induced by administration of APAP.The study was approved by the appropriate governmental institution  which granted the ethical approval for this study. The study was conducted in accordance with the principles for care and use of animals based on the Helsinki Declaration and the ARRIVE criteria.11. Briefly, after a fasting period of 12 h, the animals received 250 mg/kg of APAP  by a single i.v. injection into a tail vein. After sacrifice, diaphragm was removed quickly and further processed (see below). Alanine aminotransferase (ALT) and aspartate aminotransferase activity (UV test at 37 °C) was measured in serum .C57BL/6J mice were housed under specific pathogen free conditions  and were divided into 3 groups (each n = 8): (1) acute liver injury sacrified at 12 h (APAP12), (2) acute liver injury sacrified at 24 h (APAP24), (3) an untreated control group (CTRL). Acute liver injury was induced as described in detail beforeDiaphragm strips were longitudinally embedded in formalin and cut in 10 µm thin slides. Staining was performed using hematoxylin and eosin as routine laboratory staining. Around 50 fibers of 3 animals of each group were used for assessment of cross-sectional areas using Image J software .−1) in 5 mmol Tris·HCl and 5 mmol EDTA buffer (pH at 7.5) containing a protease inhibitor cocktail  and centrifuged at 1500×g for 10 min at 4 °C. After collection of the resulting supernatant, diaphragmatic protein content was assessed by the method of Bradford12, . Proteins were detected with Chemiluminescent Peroxidase Substrate , imaged with the Proxima 2850T imaging system  and analyzed using the TotalLab 1D software .Diaphragm tissue samples were homogenized 1:10 (weight volumeAutophagosome formation was assessed by measuring the conversion of LC3B-I to LC3B-II via immunoblotting. Proteins were separated on a 12% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. Blots were incubated overnight at 4 °C with the primary antibody  and subsequently with the appropriate secondary antibody  for 1 h at room temperature. Data were expressed as LC3B-II/LC3B-I ratio.Upstream signaling via Phosphatidyl-inositol-Kinase-3 (Pi3K) and Protein kinase B (AKT) was also measured by detecting the phosphorylated and non-phosphorylated form of Pi3K  and of Protein kinase B . Data were expressed as ratio between phosphorylated to total (non-phosphorylated) protein.13. The cleavage product of intact αII-spectrin by calpain gives a band at 150 kDa and a band at 120 kDa when cleaved by caspase-3 while intact αII-spectrin is detected at 260 kDa14. Active Caspase 3 was measured with their cleaved forms .In vivo calpain-1 and caspase-3 activities were indirectly assessed, by measuring cleavage of αII-spectrin, a specific substrate for both calpain-1 and caspase-3. After separation of the proteins on a 10% polyacrylamide gel and transfer to PVDF membrane, the blot was incubated with the primary antibody against αII-spectrin  and the suitable secondary antibody . Activities of calpain and caspase-3 were measured as the ratio between the densitometric values of their breakdown products to intact αII-spectrinThe activation of E3 ligases Atrogin and MURF-1 as markers of the ubiquitin–proteasome pathway was investigated by Western blotting using the antibodies: .15.Diaphragmatic 4-hydroxynonenal (4-HNE) was used as a marker of lipid peroxidation. After electrophoresis (7.5% polyacrylamide gel and blotting) polyclonal anti-4-HNE antibody  was used as primary antibody and a polyclonal rabbit anti-mouse  as secondary antibody. Data were expressed as a ratio between densitometric values of 4-HNE and VinculinThe p50 and p 65 subunits of NFkB were assessed by Western blotting using P50/P65: #4764 and phospho-P65: #3031, Cell-Signaling, Danvers, MA, USA.T method. The following primers were used . qPCR was run on a StepOne Plus instrument (LifeTechnologies) using Power SYBR Green Master Mix (Applied Biosystem #4368706). For messenger RNA (mRNA) analysis, inflammatory and proteolytic marker expression was normalized with ribosomal protein S7. RNA expression was analyzed by the ΔΔ CPopulation distribution was assessed with the Kolmogorov–Smirnov test. If this test showed normal distribution of data, comparisons between groups for each dependent variable were made by ANOVA, if normal distribution was not present, by a Kruskall Wallis test. If the group effect was significant, a Dunn’s multiple comparisons test was used for pairwise comparisons between all groups. Data are shown as means ± standard deviation (SD). All statistical tests are two-tailed, significance was established at p < 0.05 .Serum levels of ALT increased significantly and similarly 12 and 24 h after APAP administration compared to control  while serum levels of AST increased significantly but only 24 h after APAP administration (ctrl vs APAP 24 p < 0.001) .Caspase 3 and calpain-1 protein (measured as the amount of the specific breakdown products of α-II spectrin) were significantly decreased after 24 h after APAP intoxication vs ctrl  see Fig. .Downstream levels of Ubiquitin E3 ligases Atrogin and MURF did not differ between ctrl and the APAP treated animals , while this increase failed to reach statistical significance after 24 h see Fig. . The bloThis study showed that in mice, during the acute phase of liver injury, no activation of the proteolytic or autophagic pathways and no atrophy occurred in the diaphragm while lipid peroxidation was elevated and IL-6 was the only inflammatory marker to be upregulated inside the diaphragm. The effects on the diaphragm are discussed in detail below.10. Hepatocyte necroses are induced directly by the toxic metabolite NAPQI. Otherwise, severity of the liver injury and dimension of necrosis are affected by a massive immune cell infiltration into the liver caused by the initial damage3. Until know it is still ambiguous which part of the immune response and which immune cells respectively aggravate or attenuate liver injury at the different stages of the disease16.The APAP model to induce acute liver failure is a well-established model. In previous studies, we have proven that induction of necrosis in hepatocytes after administration of APAP leads to consistent results within groups. Analysis of immune cells and their modulation showed that the model induces “two hits” during acute liver injury generated by APAP4. Summarily, acute liver failure results in multi-organ failure (Acute liver failure)17, although depending on etiology and duration course of illness is variable. In APAP and ischemia induced ALF the acute phase will be measured in hours and we expect systemic effects during the experimental time chosen in our model.We detected significant changes of liver enzymes’ serum levels and necrotic areas inside liver tissue, pointing to a profound damage of hepatocytes. Central clinical signs of the acute liver failure are prolonged prothrombin time/INR, declined mental function, vasodilation and a systemic inflammatory response syndrome due to the high immunological impact of the liver18. Blazka and colleagues revealed an immediate response of TNFα and IL1β after APAP induced liver injury induction with systemic response19. IL-6 can even more act in several different ways in muscle and is produced during exercise20 or diaphragm overload21. Moreover, Janssen et al. demonstrated severe muscle atrophy in the diaphragm after IL-6 administration into the circulation. However, in diaphragm the influence of inflammation on its function has been under discussion: Blocking of toll-like receptor-4 in mice in a disuse model could decrease the amount of autophagic activation and IL-6 production and ameliorate the reduction of myosin heavy chain protein22. Possibly, the animals had an increase in the work of breathing due to metabolic disturbances after liver injury, which might explain this increase in IL-6 in a quasi-physiologic response to overload.Inflammatory response to acute liver injury has been described in humans and several cytokines in drug induced liver injury were elevated in serumSummarily, we could not detect a profound secondary response inside the diaphragm in the following 12 h or 24 h after liver injury induction except for enhanced levels of IL-6 that were not associated with changes in NFκB subunits or other inflammatory cytokines.5 with mitochondria and to a smaller extent oxidases functioning as major sources of ROS25. In diaphragm, the increase of ROS interacts with the activity of main proteolytic pathways caspase 3 and calpain-1 as well as the autophagic system, so that blocking ROS results in an amelioration of atrophy via autophagic and proteolytic pathways27. In our model, we could detect a significant transient increase in the formation of lipid peroxidation (4 HNE) after 12 h of APAP induced liver injury. This increase abandoned in the 24 h APAP group, which could indicate a quick removal of oxygenated structures, as we have detected in a former experiment28. In our actual study, we did not distinguish further which source (mitochondria and/or oxidases) was responsible for the increase in oxidation. Importantly, we did not found a link between this increase in lipid peroxiadation and atrophy inducing pathways, although in disuse or sepsis this has been proven27. Possibly the amount of oxidative stress did not reach the level to induce proteolytic Ca-dependent proteases or autophagy.Oxidative stress is a key factor in the pathogenesis of ICU-acquired diaphragmatic weakness31. Caspase 3 acts a key protease, in conjunction with calpain 1, to break up the sarcomeric structures and allow downstream proteasome to further degrade the proteins33. Neither mRNA levels nor protein expression of key calcium-dependent proteases were enhanced in the diaphragm despite the presence of clear liver injury. Even more, the downstream ligases MURF and MAFbx remained unaltered in the interventional groups, although these ligases are involved in the further degradation of muscle protein after break-up by caspases31 and are usually activated during muscle atrophy, inflammation or oxidative stress30. In summary, proteolytic activation in the diaphragm by acute liver injury does not seem to be present in our model.In several diseases, proteolysis is activated inside the diaphragm and modulated by different pathways: Calcium dependent protease-based proteolysis via caspase 3 and calpain 1 have been described during sepsis, disuse (VIDD) and in chronic lung disease34. Upstream, Protein kinase B (AKT) and PI3K regulate via the Forkhead-Box-O1 transcription factor the formation of autophagosome building proteins, like LC3B.Autophagy is an important proteolytic pathway targeting cellular components associated with muscular atrophy via autophagosomes, which form double layer structures of which LC3 is a core molecule35. Transformation from LCB3BI into LC3B-II indicates the activation of autophagosomes30 and Hussain and colleagues described this pathway as one major diaphragmatic pathway in disuse36 which is also active in sepsis and linked to oxidative stress30. In addition, the autophagy pathway can modulate protein synthesis through the mammalian target of rapamycin (mTOR) and serves therefore as a switch between anabolic and catabolic states37.The diaphragm is susceptible to slightest disturbances in the ratio of protein synthesis and proteolysis, so that even 12 h of disuse affect this balanceDuring liver injury as in our model, autophagy assessed by the ratio of LC3BII/LC3BI and by the upstream molecules was not activated in the diaphragm.38. Those were in this study beyond the scope.This study has, however, some limitations that should be taken into account. According to the goal of the study, 12 and 24 h were specifically chosen as time point measures after liver injury in order to address the early phase effects of liver injury on the diaphragm. We, however, acknowledge that later time point measures may be relevant to address the secondary complications reported to arise days after acute liver injury in humans. Further studies are needed to address the influence of acetaminophen induced liver injury on diaphragm dysfunction at later time points and whether known effects on lung function  may add to these changesIn contrast to our hypothesis, acute liver injury induced by APAP in mouse did not induce atrophy or activate proteolysis via calcium dependent proteases or via ubiquitin proteasome pathway, neither it did stimulate autophagy. However, it resulted in enhanced diaphragm lipid peroxidation and upregulation of IL-6 which, on long-term, might be detrimental for the diaphragm. Future research might focus on long term models of liver injury to see whether the diaphragm would be affected.Supplementary Information 1.Supplementary Information 2.


IntroductionCoronary artery bypass graft (CABG) is the most potent of surgical procedures; in this procedure, the narrowing of the coronary artery due to atherosclerotic plaque is bypassed by forming an alternate route for blood flow to the heart. There are various risk factors associated with the procedure. The aim of this study was to observe if postoperative outcomes are affected by preoperative hematocrit (hct) levels in patients.Methods This longitudinal study was conducted from April 2019 to December 2019. Eighty-two (82) participants who were to undergo CABG surgery were divided into two groups based on their preoperative hct levels. Group 1 had 42 participants with lower levels of hct (less than 35.5% for women and 38.3% for men), whereas group 2 consisted of 40 participants with normal hct levels (greater than 35.5% for women and 38.3% for men).ResultsThe results showed that participants undergoing CABG with lower than normal hct levels had increased blood loss through drainage as compared to participants who had normal hct levels . Group 1 participants also had an increased need for blood and blood product transfusion as compared to group 2 . Furthermore, the participants in group 1 had longer stays in the ICU relative to the other group .ConclusionBased on our findings, patients who undergo CABG surgery with lower than normal hct levels are at increased risk of certain complications, including excessive blood loss, need for transfusion, and increased duration of ICU stay. Therefore, preoperative hct levels should be routinely checked in patients undergoing CABG to prevent these complications. Coronary artery bypass graft (CABG) is a revascularization procedure that is used to treat coronary artery disease (CAD), which is the narrowing of the arteries supplying oxygen and nutrients to the myocardium by atherosclerotic plaques . ConsideVarious studies have been conducted in the past to determine the surgical risk factors of CABG, and it has been found that the success of the procedure depends on the improvement or elimination of these risk factors, making preoperative risk-factor assessment important in patients undergoing CABG -5. TheseThere is very limited data available on the preoperative assessment of patients undergoing CABG in the local setting. In this study, we aimed to analyze the effect of hct levels on early postoperative outcomes in patients undergoing CABG.https://clincalc.com/stats/samplesize.aspx) based on results from Pala et al. [This longitudinal study was conducted in the cardiovascular unit of a tertiary care hospital in Pakistan from April 2019 to December 2019. The sample size was calculated using an online calculator, ClinCalc . Continuous variables were presented as means and standard deviations. Binary outcomes were expressed as frequencies and percentages. The student's t-test and Pearson's chi-squared test were employed to compare the two groups. A p-value of less than 0.05 indicated that there was a significant difference between the groups and the null hypothesis was not valid.After enrollment in the study, all patients underwent phlebotomy before any intervention was given, and blood samples were sent to the laboratory to determine the hct levels. Forty-two (51.3%) patients undergoing CABG had lower levels of preoperative hct (less than 35.5% for women and 38.3% for men) and were placed in one group (group 1), and the other 40 (58.7%) participants with normal hct levels (greater than 35.5% for women and 38.3% for men) were placed in another group (group 2).Age, gender distribution, comorbidities (hypertension and type 2 diabetes) and ejection fraction were comparable between both groups. Hemoglobin (Hb) and hct were significantly lower in group 1 compared to group 2 . Group 1 participants also had an increased need for blood and blood product transfusion as compared to group 2 . Furthermore, the participants in group 1 had longer stays in the ICU relative to the other group  (Table Patient optimization prior to CABG is essential for better outcomes and early recovery. Hematocrit is a well-recognized factor with respect to pre and postoperative complications of cardiac surgery. The present study was conducted to assess the effect of hct levels on early adverse postoperative outcomes in patients undergoing CABG. In our study, postoperative drainage, the need for blood and blood product transfusion, and length of ICU stay were found to be statistically higher in patients in the low-hct group.Despite the advances in cardiovascular medicine and cardiac surgery operating techniques, morbidity and mortality are still a problem after CABG . With inMultiple studies have been conducted about this topic, and they have investigated the association of blood and blood product transfusions with outcomes in CABG patients. In a study by Surgenor et al., 36% of patients received one to two units of packed cell transfusion; among them, 43% were transfused intraoperatively, and the rest were postoperatively transfused, and the mortality rate was found to be 16% higher in patients receiving transfusion compared to those who did not receive it . A studyA low preoperative hct level is an independent risk factor for the need for a transfusion, and the mortality rate among patients is directly proportional to the need for a transfusion . SimilarIn the current study, the mortality rate was higher in group 1 compared to group 2, although the difference was statistically insignificant. Low preoperative hct levels have a negative impact on postoperative recovery, and the higher initial 30-day mortality rate in this patient group is an indication of the need for careful treatment planning. Even if the risk scoring system did not include the hct level, attention should be paid to the hct test as it will not only help the surgeon but also the patient in terms of decision-making and planning regarding the operation.The major limitation of the current study was that it was conducted at a single center; therefore, our results cannot be generalized to other centers with a higher number of patients. Another limitation was that patients were analyzed for in-hospital mortality only and were not followed up for medium and long-term results.In the present study, low preoperative hct levels were found to be associated with prolonged ICU stay among CABG patients. It was also associated with an increased need for blood transfusion and blood drainage during and after CABG, which are possible determinants of various adverse events. Therefore, detecting and treating low preoperative hct levels is critical as it may reduce unwanted postoperative complications. Moreover, we believe that preoperative hct levels should be an integral component of risk scoring systems to assess the postoperative mortality risk and to foresee hospital stay and cost.


ALBA dynamics during reproductive development in Arabidopsis at the levels of gene expression and protein localization, both under standard conditions and following heat stress. In generative tissues, ALBA proteins showed the strongest signal in mature pollen where they localized predominantly in cytoplasmic foci, particularly in regions surrounding the vegetative nucleus and sperm cells. Finally, we demonstrated the involvement of two Rpp25-like subfamily members ALBA4 and ALBA6 in RNA metabolism in mature pollen supported by their co-localization with poly(A)-binding protein 3 (PABP3). Collectively, we demonstrated the engagement of ALBA proteins in male reproductive development and the heat stress response, highlighting the involvement of ALBA4 and ALBA6 in RNA metabolism, storage and/or translational control in pollen upon heat stress. Such dynamic re-localization of ALBA proteins in a controlled, developmentally and environmentally regulated manner, likely reflects not only their redundancy but also their possible functional diversification in plants.ALBA DNA/RNA-binding proteins form an ancient family, which in eukaryotes diversified into two Rpp25-like and Rpp20-like subfamilies. In most studied model organisms, their function remains unclear, but they are usually associated with RNA metabolism, mRNA translatability and stress response. In plants, the enriched number of ALBA family members remains poorly understood. Here, we studied  ALBA-family (Acetylation lowers binding affinity) proteins belong to an ancient group of proteins found in all domains of life ,2. All mOryza sativa), nine ALBA genes were identified, showing tissue-specific expression profiles, with expression also detected in generative organs [OsALBA1, OsALBA2, OsALBA6, and OsALBA9 in the Rpp25-like subfamily, and OsALBA3, OsALBA4, OsALBA5, and OsALBA8 in the Rpp20-like subfamily [OsALBA7 has a different domain composition, not exactly falling within these two sub-families [Arabidopsis thaliana, six homologues are equally shared by the Rpp20-like  and Rpp25-like  subfamilies [ALBA genes were preferentially active in young developing tissues including root tips, cotyledons, and leaf primordia. AtALBA1 and AtALBA4 showed the highest expression in all observed tissues within Rpp20-like and Rpp25-like subfamilies while ALBA3/DAN1 was specifically expressed in flowers [In plants, ALBA homologues have been investigated in several species. Seed plant genomes underwent genome duplications and rearrangements which led to the gene duplications observed in the ALBA family ,10. In rubfamily . OsALBA7families . In Arabfamilies . Each su flowers ,13. Avai flowers , while O flowers .Plasmodium falciparum, ALBA homologues associate with factors that regulate translation repression [Leishmania infantum, temperature stress caused ALBA protein (LiAlba1/Rpp20 and LiAlba3/Rpp25) re-localization from the cytoplasm into the nucleolus and flagellum, together with PABPs [Trypanosoma brucei ALBAs were detected in association with mRNAs, exclusively in cytoplasmic stress granules following nutrient stress [ALBA genes were demonstrated following various abiotic stress and phytohormone treatments [OsALBA1, OsALBA2, OsALBA6, OsALBA7, and OsALBA8 were upregulated, while OsALBA4 was downregulated [ALBA genes in Gossypium hirsutum (cotton) leaves, roots, and stems. In leaves and roots, two genes GhALBA_4 and GhALBA_5 were significantly upregulated [AtALBA1, AtALBA2, AtALBA4, AtALBA5, and AtALBA6 enrichment was detected in the mRNA binding proteome of Arabidopsis four-day-old etiolated seedlings [Various stress conditions have been shown to cause changes in ALBA behavior. In pression . In Leisth PABPs . Similart stress ,8. In plt stress ,14,15. Ieatments . Most geegulated . SimilarALBA expression by various abiotic stresses, we studied the expression dynamics of all ALBA genes in Arabidopsis generative tissues and investigated the response of a subset of them to heat stress.Increased temperatures often has a harmful effect on plant reproduction and fertility. Reproductive development is particularly sensitive to heat stress treatment . The malArabidopsis ALBA family is well conserved and comprises six homologs. The AtALBA genes form two subfamilies (ALBA1 (At1g29250), ALBA2 (At2g34160) and ALBA3 (At3g04620)) possessing only an ALBA domain, whereas the Rpp25-like subfamily contains longer genes (ALBA4 (At1g76010), ALBA5 (At1g20220) and ALBA6 (At3g07030)) carrying an ALBA domain and a RGG rich carboxyterminal extension  with different untranslated regions (5’UTR and 3´UTR). ALBA6 gene on the other hand produces four splice variants ALBA6-1 (At3g07030.1), ALBA6-3 (At3g07030.3), ALBA6-4 (At3g07030.4), and ALBA6-5 (At3g07030.5). These sequences were analyzed and their relations compared using a maximum likelihood unrooted tree construction  and in sperm cells  or sharp between bicellular and tricellular pollen (ALBA1 and ALBA4). In contrast to these genes, ALBA3/DAN1 expression, activated by the DUO1 transcription factor [ALBA2-ALBA6 was detected in sperm cells at the mature pollen stage. This enhancement was particularly strong for ALBA3/DAN1.Following the analyses of the rm cells C. With tn factor , shows aALBA expression more closely, ALBA promoter regions were fused to the β-glucuronidase (GUS) reporter gene and their activity documented in transformed (ALBA1-6) and untransformed Col-0 plants. T1 generation plants were screened for GUS expression with at least two independent lines with a representative and stable GUS expression pattern selected per construct. During reproductive development, various patterns of expression were found within the ALBA family. We observed similarities between both subfamilies. In each subfamily, there was one dominantly expressed gene (ALBA1—Rpp20-like and ALBA4—Rpp25-like) with the remaining two genes showing weaker expression.To investigate ALBA1 promoter is highly active in inflorescence stems, flower buds, and developing and maturing flowers  and disappeared towards the middle part. Moreover, ALBA4 activity becomes stronger during pistil maturation and its conversion into a silique, where it unequally expands from distal areas towards the center with the only signal connection in septum  and sperm cells (SC) . In the Of them, ALBA3-GFP appeared to preferentially localize in the sperm cells around sperm cell nuclei. In addition to this preferential localization in proximity to the MGU, both these proteins also accumulated in distinct foci of variable size in the VC cytoplasm.The two remaining Rpp20-like subfamily members, ALBA1-GFP and ALBA2-GFP, were less abundant. They accumulated preferentially in round foci attached to VN and in the large, elongated particles in close proximity to SC nuclei,  or moderate (42 °C for 1 h) heat stress, their inflorescences collected (1 h or 24 h post-treatment to distinguish early and late heat stress response) for RNA isolation and RT-qPCR subsequently carried out. Melting curve analysis consistently demonstrated a single homogenous melting peak for all primer sets (GAPC1 (At3g04120) and EIF1a4 (At5g60390) as reference genes for the experiments and evaluated the data  1 h and 24 h after the application of 37 °C heat stress (HS)  revealed a statistically significant decrease after the treatment. Interestingly, ALBA expression increased 24 h following HS  in ALBA expression  . All ALBcontrols A. Howeveowing HS B. The appression . HoweverproALBA6::GFP-GUS-transformed pollen was not affected by heat-stress and served as a control . Mature pollen was harvested 1 h or 24 h after HS to distinguish the early and late response, respectively. As under standard conditions , the stu control .All three Rpp20-like subfamily members showed different stress-induced localization patterns. ALBA1-GFP, less abundant in mature pollen under control conditions, increased in abundance shortly after 37 °C treatment, and accumulated around MGU in several large cytoplasmic particles, whereas the GFP signal was less abundant in pollen VC collected 24 h after the treatment A1–B2. HeThe heat stress response of the two less abundant Rpp25-like subfamily members, ALBA4-GFP and ALBA5-GFP, was less dramatic. Neither showed enrichment around MGU under any conditions. Within the subfamily, a stronger signal accumulation in cytoplasmic foci was captured in ALBA4-GFP pollen after the 37 °C treatment A7–B8. A ALBA3-GFP and ALBA6-GFP, being the most abundant ALBAs in untreated mature pollen , both acp < 0.001) enhanced both ALBA4-GFP and ALBA6-GFP protein accumulation in cytoplasmic foci, which were still visible, albeit more dispersed, 24 h after HS.Based on these results, we aimed to quantify changes in ALBA protein localization with respect to particular foci formation under standard and HS conditions. Strong protein re-localization upon HS focused our attention to Rpp25-like subfamily proteins. We selected two proteins, ALBA4-GFP and ALBA6-GFP for precise quantification of HS-induced protein clustering. The degree of protein clustering was quantified by the coefficient of variation (CV) reflecting fluctuations in fluorescence signal intensities. CV was estimated in untreated pollen and after the exposure of flowering plants to 37 °C for 3 h, both 1 h and 24 h after the treatment . Only opArabidopsis, PABP3, and PABP5 show an organ-specific expression pattern in floral organs, including the male gametophyte [PABP3 is natively expressed in tapetum and pollen [ALBA family proteins are known to be associated with nucleic acids ,3,4,5,7.etophyte ,23. PABPd pollen . Since tPABP3-RFP localizes predominantly in cytoplasmic foci equally distributed in the VC cytoplasm, enriched in the MGU region . A detaiDue to similarities in localization of ALBA4-GFP, ALBA6-GFP, and PABP3-RFP, we decided to analyze their co-localization. To this effect, we used plants co-expressing ALBA4-GFP or ALBA6-GFP and PABP3-RFP. Our analysis revealed a strong signal overlap between ALBA4-GFP and PABP3-RFP in the VC cytoplasm . The co-Pollen of heterozygous ALBA6-GFP/PABP3-RFP plants was used for further co-localization analysis. The ALBA6-GFP signal is strongly enriched around SC nuclei where it rarely overlaps with the red signal of the mRNA marker . On the contrary, moderate HS (42 °C) had no significant effect on ALBA expression. These findings suggest that ALBA genes are regulated in a temperature-dependent manner, and are likely involved in mild temperature stress response in Arabidopsis. As such, ALBA genes could act as thermomemory-associated genes [ALBA expression led us to investigate ALBA-GFP localization in response to heat stress in mature pollen. The application of mild HS (37 °C for 3 h) affected ALBA-GFP localization, especially for ALBA4 and ALBA6, the two more divergent Rpp25-like subfamily members, with the most stable signal localization pattern. Similar stress-triggered re-localization of ALBA proteins between the cytoplasm and cytoplasmic foci were reported in Trypanosoma and Plasmodium [In rice, stresses . In thisstresses . We quaned genes  in the mmild HS 3 °C. On tasmodium .Leishmania [Plasmodium [Trypanosoma [Arabidopsis ALBAs, including ALBA4 and ALBA6 [Trypanosoma [Toxoplasma [Leishmania [We further traced mRNA distribution in pollen by RNA-binding protein PABP3-RFP ,33 with ishmania , Plasmodasmodium  and Tryppanosoma ,34 in stpanosoma . Therefond ALBA6 , their cpanosoma , Toxoplaxoplasma , and Leiishmania ,19,31,36Collectively we demonstrated the involvement of ALBA-family proteins in male reproductive development and in the heat stress response. Moreover, we suggest that ALBA4 and ALBA6 are implicated in RNA metabolism and storage in pollen upon heat stress. These two members  can dynamically re-localize between various types of mRNA-containing granules within the cytoplasmic mRNPs continuum in a controlled, developmentally and environmentally regulated manner. Such regulation then reflects not only their redundancy but also their possible functional diversification in plants. https://www.arabidopsis.org/). Motif analysis was performed using the MEME suite 5.2.0 [All DNA, cDNA, and protein sequences used were obtained from TAIR  [Transcriptomics data for gene expression analysis were obtained from the CoNekT online Database (t.tools) .ALBA1-ALBA5 containing coding regions and their native promoters were amplified from genomic DNA using specific primers (ALBA4 (genomic DNA) and ALBA6 (ALBA6-4 isoform amplified from pollen cDNA) gene fragments were amplified from pollen cDNA and cloned using the GoldenBraid 3.0 system into the pDGB3 ω2 destination binary vector [ALBA4 and ALBA6 coding regions were domesticated into pUPD2 entry vector [For promoter fusion constructs, proALBA::GUS-GFP, native promoter sequence fragments of all ALBA genes were amplified from genomic DNA using specific primers  transformation. Stable transgenic plants were obtained by floral dipping [For the co-localization of ALBA4-YFP and ALBA6-mCherry, we used GoldenBraid cloning to combine both transcriptional units into a single destination vector ,47. All  dipping . Seeds oArabidopsis stable line expressing a tagged version of PABP3, the whole genomic sequence encompassing PABP3 (AT1G22760) (starting from position—1884 from ATG to the last nucleotide before stop codon and including introns and 5’-UTR) was PCR amplified using primers eb1 & eb2  were germinated on 0.5× Murashige and Skoog (MS) medium containing 5% (w/v) Sucrose, 25% (w/v) MES and 0.8% (w/v) Agar [/v) Agar , and tra/v) Agar . Transfo20 primer. RT-qPCR measurements were obtained using GoTaq Q-PCR Master Mix  on a LightCycler 480  with gene specific primers (GAPC1 (At3g04120) and EIF1a4 (At5g60390) expression levels and statistics calculated using R [Total RNA was extracted from inflorescences collected 1 h and 24 h after heat stress application (37 °C for 3 h and 42 °C for 1 h) using the RNeasy Mini Kit . Four biological replicates (inflorescences) were used for each experiment. RNA quantity and quality (purity) was determined using NanoDrop One . RNA quality was verified by electrophoresis in a 2% agarose gel. All samples were treated by RQ1 RNase-Free DNase . Reverse transcription (RT) was performed for 1 h at 42 °C using ImProm-II™ Reverse Transcription System  with an oligo-d(T) using R . The datproALBA::GFP-GUS transgenic plants were stained for GUS activity with a solution containing 50 mM sodium phosphate buffer, pH 7, 0.2% Triton X-100, 1 mM X-Gluc  and 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide or 2.5 mM potassium ferrocyanide, 2.5 mM potassium ferricyanide. Samples were vacuum-infiltrated for 10 min and stained at 37 °C. The staining was fixed in 99% ethanol for 24 h after which it was replaced by 70% ethanol at 4 °C.The GUS staining protocol was adapted to the individual samples, with two solutions used for the final staining using the appropriate time in between 7 and 24 h. Inflorescences of T2 generation Pollen from all samples was collected and stained with 0.8 µg/mL DAPI  in GUS buffer for 15 min (mature pollen) or 1 h  . StainedproALBA::GUS-GFP inflorescences, stamens with unopened and opened anthers, pistils from unopened and opened flowers and young siliques were collected and captured by a Zeiss stereomicroscope . Nectaries, ovules, seeds, microspores, bicellular and mature pollen grains were collected and captured by a widefield fluorescence microscope Zeiss Axioimager .Fixed Pollen grains of transgenic plants carrying ALBA-GFP and PABP3-RFP, ALBA6-mCherry and ALBA4-YFP and GUS-GFP were collected on a slide containing the DAPI staining solution and imaged under the inverted confocal laser scanning microscope Zeiss LSM880  equipped with an Airyscan detector and Plan-Apochromat 100×/1.46 Oil objective. For excitation of DAPI, GFP, RFP/mCherry laser lines 405 nm, 488 nm and 561 nm were used in a sequential scanning setup. Within an experiment, all images were acquired using adjusted settings reflecting various signal intensities of the individual fusion proteins. Airyscan Processing of raw files was performed in ZEN black software .Microscopy data were analyzed in the ZEN blue 2.5 software —Maximum intensity projection, 3D-view, and colocalization analysis. Coefficient of variation (CV) was estimated as described in  and modiPearson’s correlation coefficients were calculated in Zen blue SW from each thresholded cross-section of the z-stack. Average values from both analyses were depicted as box plots, their statistical significance was calculated using One Way ANNOVA followed by Pairwise Multiple Comparison Procedures (Dunns method) in Sigma Plot .


We analyzed the radiolarian assemblages of 59 surface sediment samples collected from the Yellow Sea and East China Sea of the northwestern Pacific. In the study region, the Kuroshio Current and its derivative branches exerted a crucial impact on radiolarian composition and distribution. Radiolarians in the Yellow Sea shelf showed a quite low abundance as no tests were found in 15 of 25 Yellow Sea samples. Radiolarians in the East China Sea shelf could be divided into three regional groups: the East China Sea north region group, the East China Sea middle region group, and the East China Sea south region group. The results of the redundancy analysis suggested that the Sea Surface Temperature and Sea Surface Salinity were primary environmental variables explaining species-environment relationship. The gradients of temperature, salinity, and species diversity reflect the powerful influence of the Kuroshio Current in the study area. Polycystine Radiolaria (hereafter Radiolaria), with a high diversity of 1192 Cenozoic fossil to Recent species, are a crucial group of marine planktonic protists . Living The East China Sea (ECS) and Yellow Sea (YS) are marginal seas of the northwestern Pacific . The twoThe Kuroshio Current originates from the Philippine Sea, flows through the ECS, and afterwards forms the Kuroshio Extension . The KurIn the ECS shelf region’s summer , the KurIn the YS shelf region’s summer , the YelThe radiolarian assemblages in surface sediments have been investigated in the ECS whereas there are few reports in the YS. These reports cover the ECS including the Okinawa Trough  and contDictyocoryne profunda Ehrenberg, Dictyocoryne truncatum (Ehrenberg), Dictyocoryne bandaicum (Harting) were combined as Dictyocoryne group. Photographs of some radiolarians encountered in this study are exhibited in The surface sediments were collected at 59 sites  in the YGrain size analysis of the surface sediments was conducted with a Laser Diffraction Particle Size Analyzer . The data were used to categorise grain size classes as clay (1–4 µm), silt (4–63 µm) and sand (63–500 µm), and to determine different sediment types according to the Folk classification . In addia and particulate organic carbon with a 9 km resolution for the period of 1997 to 2010 were obtained from https://oceancolor.gsfc.nasa.gov/l3/. The values of the environmental variables mentioned above for each surface sediment site were estimated by linear interpolation. These values, together with depth, are shown in The values of annual temperature (SST), salinity (SSS), oxygen, phosphate, nitrate, and silicate of sea surface with a 0.5° resolution for the period of 1930 to 2009 were derived from the CARS2009 dataset . The seaThe minimum number of specimens counted in each sample is customarily 300. However, low radiolarian concentrations are frequent in the shelf type sediments comprised mainly of terrigenous sources . Given s−1) and the diversity indices, including the species number (S), Shannon-Wiener’s index (H′ (loge)). To ensure a creditable estimate of diversity indices, which may be biased by different counting numbers, the specimens of radiolarians in each sample were randomly subsampled and normalized to the equal size of 100 tests by using rrarefy function in vegan package in R program. For each site, S and H′ of sample containing all tests and subsample containing 100 tests were calculated.We calculated the absolute abundance (tests.(100 g)Relative abundance (%) of each radiolarian taxon was also calculated. Then the hierarchical cluster analysis with group-average linking was applied to analyze the variations of radiolarian assemblage among different regions. The percentage data of the relative abundance was transformed by square root for normalize the dataset. Afterwards, triangular resemblance matrix was constructed based on the Bray-Curtis similarity . Analysia, silicate, particulate organic carbon, oxygen, depth, nitrate, and silt percentage were removed from the RDA model step by step, in order to avoid collinearity  was applied to determine the character of the species data. The gradient length of the first DCA axis was 1.773 < 3, suggesting that redundancy analysis  was more suitable than canonical correspondence analysis  . RDA wasinearity . FinallyCorrelation analysis was employed to investigate the relationship between the dominant radiolarian taxa and significant environmental variables.The diversity indices calculation, cluster analysis, ANOSIM, and SIMPER were performed by PRIMER 6.0. Correlation analysis was performed by SPSS 20. DCA and RDA were conducted by CANOCO 4.5. The detailed description of the statistical methods, including the cluster analysis, DCA, and RDA are in A total of 137 radiolarian taxa were identified from the surface sediments of study area, including 75 genera, 14 families, and three orders The raw radiolarian counting data are in −1) > ECSSR (1776 tests. (100 g)−1) > ECSNR (500 tests. (100 g)−1) > YSR (8 tests. (100 g)−1). The distribution pattern of species number (Tetrapyle octacantha group Müller (55.6%), Didymocyrtis tetrathalamus (Haeckel) (7.5%), Dictyocoryne group (3.7%), Spongaster tetras Ehrenberg (2.5%), Stylodictya multispina Haeckel (2.2%), Spongodiscus resurgens Ehrenberg (2.2%), Zygocircus piscicaudatus Popofsky (2.1%), Phorticium pylonium Haeckel (2.0%), and Euchitonia furcata Ehrenberg (1.8%).Radiolarian abundance in surface sediments varied greatly in study area , showings number  was simi−1 to 91 tests. (100 g)−1, and species number ranged from 0 to 12. Tetrapyle octacantha (17.4%), Spongodiscus sp. (10.9%), Didymocyrtis tetrathalamus (9.1%), Acrosphaera spinosa (6.1%), and P. pylonium (6.1%) were the five most abundant species taxa in the YS, constituting 49.7% of the total assemblages.In general, radiolarians showed a quite low abundance value in the YS, as no tests were found in 15 samples . For thep = 0.001). Diversity indices, including S and H′, displayed an overall ranking of ECSSR > ECSMR > ECSNR both in samples  and subsamples .As can be seen in R = 0.769, p = 0.001).Cluster analysis based on the relative abundance classified all but one site into three regional groups at the 60% Bray-Curtis similarity level, including the ECSNR group, ECSMR group and ECSSR group . The sigTetrapyle octacantha, Didymocyrtis tetrathalamus, and Spongodiscus resurgens dominated in the ECSNR group, with contribution of 41.70%, 9.79%, and 8.89%, respectively. The radiolarian taxa, including T. octacantha, Didymocyrtis tetrathalamus, Dictyocoryne group, Stylodictya multispina, and Spongodiscus resurgens, contributed most to the ECSMR group. The dominant species in the ECSSR group were composed of T. octacantha, Didymocyrtis tetrathalamus, Dictyocoryne group, Spongaster tetras, Z. piscicaudatus, P. pylonium, Stylodictya multispina, and E. furcata.The dominant species in each regional group were identified by SIMPER analysis with a cut-off of 50% . TetrapyThe first two RDA axes explained 39.9%  of the species variance, and 86.5% of the species-environment relation variance . ForwardThe RDA plot showed a clear distribution pattern of regional samples . The ECSSpongaster tetras, Dictyocoryne group, Z. piscicaudatus, E. furcata, P. pylonium and Stylodictya multispina, were related to high SST, while showed little relationship with SSS. Didymocyrtis tetrathalamus was positively related to SST and SSS. Tetrapyle octacantha showed a preference for high SSS. Additionally, Spongodiscus resurgens was adapted to relatively low SST and SSS.The dominant species identified by the SIMPER analysis  are dispGenerally, the number of the radiolarian tests in continental shelf sediments of the ECS and YS is several orders of magnitude lower than that of the adjacent Okinawa trough . FirstlySpongodiscus sp., were reported as typical warm species  and ECSNR group (0.06%). Members of Pterocorys are shallow-water dwellers, as reported by Pterocorys campanula frequently occurs and dominates in the South China Sea, whereas there are no reports of the dominance of P. campanula in the sediment samples of the ECS  and salinity (33.3–34.2 psu) . Some of furcata , Fig. 8. furcata . The rel the ECS . The higT. octacantha, Didymocyrtis tetrathalamus, Dictyocoryne group, Stylodictya multispina and Spongodiscus resurgens . After removing 3000-1, no significant correlation existed between SSS and the relative abundance of T. octacantha . Tetrapyle octacantha, as the most abundant taxon in the subtropical area  The radiolarian abundance in the YS was quite low, and no radiolarians were detected in 15 of 25 YS sites.(2) The radiolarian abundance and diversity in the ECS, which is controlled by the Kuroshio warm water, was much higher. Based on the cluster analysis, the radiolarian assemblages in the ECS could be divided into three regional groups, namely the ECSNR group, the ECSMR group and the ECSSR group.T. octacantha, Didymocyrtis tetrathalamus, and Spongodiscus resurgens.a. The ECSNR group was chiefly impacted by the Changjiang Diluted Water and Kuroshio Current, with dominant species of T. octacantha, Didymocyrtis tetrathalamus, Dictyocoryne group, Stylodictya multispina, and Spongodiscus resurgens.b. The ECSMR group was controlled by the Kuroshio Current, TWC and Changjiang Diluted Water. Species contributed most to this group included T. octacantha, Didymocyrtis tetrathalamus, Dictyocoryne group, Spongaster tetras, Z. piscicaudatus, P. pylonium, Stylodictya multispina, and Euchitonia furcata.c. The ECSSR group was affected by the Kuroshio Current and TWC, in which the TWC occupies major status. The dominant species in this group were composed of (3) The RDA results indicated that SST and SSS were main environmental variables that influenced the radiolarian composition in the ECS shelf.10.7717/peerj.9976/supp-1Table S1Click here for additional data file.10.7717/peerj.9976/supp-2Table S2Click here for additional data file.10.7717/peerj.9976/supp-3Supplemental Information 1Click here for additional data file.


Distal gastrectomy with lymph node dissection, a standard operative technique for gastric cancer treatment, is safely performed because the stomach has a rich vascular supply. Gastric remnant necrosis caused by cholesterol crystal embolization following distal gastrectomy has not been described previously. We report a case of gastric remnant necrosis in a patient with cholesterol crystal embolization.A 70-year-old man with a history of cholesterol crystal embolization presented to our surgery department with complaints of anorexia and dysphasia. He was diagnosed with gastric cancer invading the pyloric antrum and underwent distal gastrectomy with Billroth 2 reconstruction. On postoperative day 11, he developed abdominal pain without fever. Emergency laparotomy revealed that most parts of the remnant stomach were necrosed. Total gastrectomy with Roux-en-Y reconstruction and abscess drainage were performed. After surgery, anastomotic leakage occurred and was treated conservatively. However, the superior pancreaticoduodenal artery aneurysm suddenly ruptured and he expired.Gastric remnant necrosis after distal gastrectomy can be a gastrointestinal presentation of cholesterol crystal embolization. Perioperative/intraoperative risk assessments such as preventive total gastrectomy or intraoperative assessment with indocyanine green fluorescence angiography may be desirable to avoid this complication. The stomach has a rich vascular supply provided by a collateral arterial plexus . The safCholesterol crystal embolization (CCE) is a rare manifestation of atherosclerotic disease . CholestHere, we report the case of a patient who developed gastric remnant necrosis after a distal gastrectomy that was caused by CCE. Our aim in presenting this report is to raise the awareness of the possible risk factors for CCE and preventive measures that may be taken to avoid this complication.A 70-year-old man presented with anorexia and dysphagia. His past medical history was notable for type 2 diabetes, hypertension, chronic kidney disease requiring hemodialysis, and CCE. The CCE was triggered by a percutaneous coronary intervention in the context of myocardial infarction 7 years earlier. He had no history of smoking or alcohol abuse. Physical examination revealed cyanosis of both feet with dry gangrene of the distal toes but no livedo reticularis. The initial laboratory test results revealed a serum hemoglobin level of 8.0 g/dL, total white cell count of 9850/μL with 5.9% eosinophils , serum carcinoembryonic antigen level of 3.2 ng/dL, and carbohydrate antigen 19–9 level of 15.3 ng/dL. Endoscopic examination revealed a type 4 tumor (Borrmann classification) circumferentially located, with invasion of the pyloric ring  and an increased C-reactive protein level (21.13 mg/dL). Abdominal CT demonstrated a gastric wall discontinuity of the remnant stomach with fluid collection in the left subphrenic space. Gastric remnant necrosis was suspected, and an emergency laparotomy was performed.Laparotomy revealed a necrotic gastric remnant. Most parts of the stomach, except for the greater curvature around the spleen, were completely necrosed Fig. . Total gWe described a case where gastric remnant necrosis developed after a distal gastrectomy. To the best of our knowledge, this is the first report of gastric remnant necrosis after gastrectomy caused by CCE. This case is instructive because 1) gastric necrosis can be a presentation of CCE, in addition to kidney and skin involvement, and 2) the mortality of gastric remnant necrosis after gastrectomy is very high; therefore, it is essential to manage this risk before and during surgery.CCE, also referred to as atheroembolism or cholesterol embolization syndrome, is a rare manifestation of atherosclerotic disease . It occuCCE affects multiple organs, especially those vulnerable to low blood perfusion such as the skin, kidneys, gastrointestinal system, and brain . The gasThe stomach has a rich blood supply and an extensive submucosal plexus. Gastric necrosis in patients with CCE may not have been reported because of the stomach’s abundant blood flow. Nevertheless, in our case, transient reduction of blood supply into the gastric remnant occurred during surgery, eventually leading to a postoperative catastrophe. The mortality associated with gastric remnant necrosis has been reported to be as high as 70% .The main arterial feeders of the remnant stomach after distal gastrectomy with D2 lymph node dissection are the short gastric artery, the posterior gastric artery, and the left subphrenic artery. When these arteries are preserved, blood flow to the gastric remnant is usually sufficient. However, this was not the case here. Interruption of the blood flow to the gastric remnant causes necrosis. Several reported cases have occurred secondary to splenic infarction . HoweverBecause the mortality of gastric remnant necrosis is very high, prevention is essential. CT angiography can be used to assess preoperative perfusion from the large vessels of the gastric walls . HoweverIn conclusion, we encountered a case of gastric remnant necrosis caused by CCE after distal gastrectomy. Gastric necrosis can be a gastrointestinal presentation of CCE, and perioperative/intraoperative risk assessments may help prevent this complication.


The COVID-19 pandemic and the measures adopted are having a profound impact on a major goal of public healthcare systems: universal access to health services. The objective is to synthesize the available knowledge on access to health care for non-COVID-19 conditions and to identify knowledge gaps. A scoping review was conducted searching different databases  for original articles published between December 2019 and September 2021. A total of 53 articles were selected and analyzed using the Aday and Andersen framework as a guide. Of these, 37 analyzed changes in levels of use of health services, 15 focused on the influencing factors and barriers to access, and 1 studied both aspects. Most focused on specific diseases and the early stages of the pandemic, based on a review of records. Analyses of the impact on primary care services’ use, unmet needs or inequalities in access were scarce. A generalized reduction in the use of health services was described. The most frequent access barrier described for non-COVID-19 conditions related to the services was a lack of resources, while barriers related to the population were predisposing  and enabling characteristics . In conclusion, our results show a general reduction in services’ use in the early stages of the pandemic, as well as new barriers to access and the exacerbation of existing ones. In view of these results, more studies are required on the subsequent stages of the pandemic, to shed more light on the factors that have influenced access and the pandemic’s impact on equity of access. The pandemic due to coronavirus SARS-CoV-2 COVID-19), a novel virus initially reported in December 2019 9, a nove,4,5,6.In this regard, some strategies taken to combat soaring COVID-19 infection rates may have negatively affected access to health services for other conditions. Firstly, at the health services level, one of the most influential measures was the classification of services as essential or non-essential, following WHO guidelines, which allowed resources to be redirected to the pandemic response. However, this has also caused cancellations or delays in elective and non-urgent procedures ,5,6,7, dAnother indirect effect of the pandemic, the economic crisis stemming from the substantial curtailment of economic activity, and the ensuing rise in unemployment and loss of household income, have aggravated associated access barriers , thereby accentuating existing inequalities in access, as studies on previous economic crises have shown . AlthougIn short, as in other epidemics and previous outbreaks, the health repercussions of the current pandemic are not confined solely to COVID-19 infection and mortality. They also include indirect negative effects on healthcare access and on the quality of curative and preventive care provided for other conditions, and the exacerbation of difficulties and barriers related to socioeconomic factors ,16,17. TWhile a plethora of scientific papers have been published on COVID-19 since the start of the pandemic, studies on its impact on access to health services have not been so plentiful. A few literature reviews have been found that summarize changes in health services due to the pandemic, focusing mainly on the adoption of telemedicine ,30,31,32Access to care involves many highly interdependent factors and stakeholders at play . This stThe aim of this article is to synthesize the knowledge accrued from the onset of the pandemic in March 2020 through to September 2021 on the impact of the COVID-19 pandemic on access to health services  for non-COVID-related conditions, and to identify knowledge gaps on these subjects.A scoping review of the scientific literature  was carrIn our bibliographic search, several digital databases were consulted to minimize the risk of overlooking any relevant studies: Medline, Google Scholar, SCiELO, and Web of Science. The search was performed over two separate periods: 22 January 2021–31 March 2021 and 22 September 2021–10 October 2021. In the Medline database, using a thesaurus, MeSH terms were employed for: (a) COVID-19: “Coronavirus Infections”, “Coronavirus”, “COVID-19”, “SARS-CoV-2”; (b) access to health services: “Health services availability”, “Health services needs and demand”, “Healthcare disparities”, “Needs assessment, healthcare”, “Health policy”, “Equipment and Supplies Utilization”, “Facilities and Services Utilization” —that used qualitative and/or quantitative methods and analyzed or described changes in access to health services in the context of the COVID-19 pandemic. The initial selection of studies to review was performed through title and abstract screening. Where there was any doubt about whether to include a study, this was discussed with another researcher in the team. Following Aday and Andersen’s framework ,40, the From the search results, 242 articles were identified for title and abstract screening, and 95 for full-text review. A total of 53 articles met the inclusion criteria for analysis .Of the 53 articles selected, 37 analyzed changes in realized access applying quantitative methods ,80,81,82With regard to the type of service, of the studies on realized access, 5 focused on health services in general ,48,49,50In terms of geographical area, 19 studies were conducted in European countries ,90,92,95With regard to the period of analysis, 38 of the selected studies were conducted over the first months of the pandemic (February to June 2020) ,92,93,98Below is a summary of results found regarding changes in realized access and potential access, following the Aday and Andersen theoretical framework ,40.Of the 38 studies that analyzed changes in realized access ,81,82,98By type of service analyzed, studies focusing on the health services in general ,47,49,50In the studies that analyzed influencing factors in the use of services in the context of the pandemic, the probability of lower utilization levels was associated with different factors. With regard to predisposing characteristics of the population, women ,79 and eThe 16 studies that analyzed potential access ,96,97,98Twelve studies described changes related to characteristics of the services ,95,97,98Fourteen studies identified barriers related to population characteristics ,96,97,98The impact of the COVID-19 pandemic has been felt worldwide in many different spheres of society, but especially in access to health services for unrelated conditions. There is now a pressing need to evaluate the changes that have arisen in this regard, and their implications for equity of access and the resilience of national health systems to future pandemics. This is, to the best of our knowledge, the first scoping review to offer a general overview of the subject, taking in the current evidence and highlighting the areas that will require further research in future studies.Most of the studies included in the analysis describe a lower level of health services’ utilization and changes in potential access, as preexisting barriers have intensified and new ones have arisen. However, while investigations into the impact of the COVID-19 pandemic are still ongoing, the results of this review show that the studies conducted to date are limited in terms of scope and methodology, and that they are mainly centered on analyzing the impact on the use of services for specific diseases or population groups during the first stages of the pandemic, with a particular focus on secondary care.Studies on the use of health services in general are very scarce, as are those on access to primary care, which in many countries has been the most overwhelmed care level due to having to take on more pandemic-related care duties . Likewise, there is a considerable lack of evidence so far on how changes have taken place over the course of the different waves of COVID-19, and according to geographical context. Although some studies with longer timeframes—to the end of 2020—have already described new drops in health services’ activity following brief periods of recovery ,63,71, fIt is important to bear in mind that access to care involves multiple interdependent factors and numerous actors. However, the results of this review show that, so far, there are almost no published studies with a wide scope using mixed methods, and that the qualitative studies available to date are still limited in both number and perspective. In regard to the latter, few studies include, in addition to that of users, other viewpoints such as that of the health professionals or managers involved in the process of taking measures or adopting practices that influenced access to care. Furthermore, the population groups selected for study were generally sufferers of a specific condition or of vulnerable status. The number of qualitative studies is probably limited due to the complexity of their development in the pandemic context, in terms of time, resources, and restrictions imposed by the mitigation measures. Approaches that combine multiple sources of evidence and different perspectives are needed to shed more light on the factors and actors that have influenced access.With regard to our main findings on the reduction in the use of services, this may be related to health systems prioritizing their response to the public health emergency, which differed according to context ,99,100. Going into more detail, the reductions in access to services described appear to have brought with them an increase in medical complications and emergencies, especially in elective procedures ,77,101, In this regard, some studies highlight the difficulties involved in maintaining normal levels of activity in certain services, even in some classed as essential, such as maternal health, oncology, or mental health ,94,97,98The few studies that analyze the influencing factors on the lower utilization of services mainly highlight a greater downturn in use among low-income users, those with limited healthcare coverage, and ethnic minorities ,74, as wAs regards the changes in potential access detected in this review, the results indicate both the exacerbation of existing barriers, related above all to structural difficulties and situations of vulnerability, and the creation of new ones. In terms of existing barriers accentuated by the pandemic, some studies reported a shortage of resources in the services to meet all the incoming healthcare requests ,95,97,98In addition, new barriers may have been created as a result of adopting alternatives to face-to-face visits  and changes in attitudes and beliefs developed as a result of the pandemic. In this regard, the use of online consultation has grown as a way to mitigate difficulties in access , but notLikewise, one of the individual characteristics that has been most influential in terms of new barriers created by the pandemic is fear of contagion ,94,95,97While it is true that various studies have identified both new barriers and the exacerbation of existing ones, the behavior of individuals in this type of public health emergency requires more in-depth analysis in order to steer the design of interventions to help counter these barriers, such as public health information campaigns or specific measures for vulnerable populations.On a final note, this review has several limitations. In the first place the nature of the studies covered varies greatly, in terms of methodology , and of geographical areas and health systems analyzed; thus, it is not possible to draw comparisons between them. Secondly, articles that analyzed access to services due to COVID-19 in addition to other illnesses were excluded from the study. This decision was made in order to rule out bias towards activity and resources destined to the treatment of other diseases. Third, no studies focusing on the impact of the pandemic control measures on access to health services were found, probably due to the limited terms used in our search to capture this area. Moreover, it is also possibly due to the difficulties involved in distinguishing the impact of the measures from other effects of the pandemic. Finally, some articles may not have been considered on being published in other languages , so this analysis may have excluded relevant information and failed to consider certain contexts. In spite of these limitations, this is the first study to address changes in access from a global perspective, with a view to shedding light on gaps in knowledge that will require further research in the future.This review analyzed studies that reported changes in health services’ utilization, and the factors that influenced the use of services for non-COVID-19 conditions, during the COVID-19 pandemic. Results vary according to the context analyzed, although, in general terms, they reflect the same trend, describing a general reduction in the use of health services, the exacerbation of preexisting barriers, and the emergence of new ones. This scoping review has shown that most studies conducted to date are limited in terms of scope and methodology and are centered mainly on the impact on specific conditions or population groups during the early stages of the pandemic, focusing mostly on secondary care. Furthermore, a significant gap in knowledge was detected on whether the services have recovered to pre-pandemic levels of care use and, if not, in which areas and for what reasons. Future studies should go into greater depth on the pandemic-related changes that have influenced access to health services , according to context and over the course of the different stages of the pandemic. In any case, as an ongoing phenomenon, the real impact of the COVID-19 pandemic is yet to be determined.


Aspergillus niger SAP2211 (accession number: MK503444.1) as an antimicrobial, anti-cancerous and anti-angiogenic agent. The synthesized Ag-NPs were characterized following UV–vis, FTIR, XRD, SEM and TEM, and were found to possess bactericidal activity against the selected pathogenic microbes, such as Staphylococcus aureus, Escherichia coli, and Salmonella typhi. Further, we evaluated cytotoxicity effect of this biogenic Ag-NPs using MMT assay on normal cardio myoblast (H9C2) and cancerous human cervical carcinoma (HeLa) cells. Doxorubicin used as positive control. This Ag-NPs have shown trivial cytotoxicity at the IC50 concentration on normal cells (IC50 = 47.17 µg/ml) over the cancer cells (IC50 = 8.609 µg/ml) with nearly 7 fold difference, indicating it as a selective anti-cancerous agent in contrast to standard drug doxorubicin (IC50 = 6.338 µg/ml). Further in-vitro assessment of wound healing capability by scratch wound healing assay, invasion by transwell matrigel invasion assay, and apoptosis via DAPI and annexin V-FITC assays were studied in HeLa cells. Synthesized biogenic Ag-NPs have shown to be anti-angiogenic in nature, which was established by in-vivo chick chorioallantois membrane assay. Overall, in vitro studies revealed that biogenic Ag-NPs positively inhibited migration, invasion, and induced apoptosis, and in-vivo CAM assay revealed that intercapillary network was reduced and the angiogenesis was inhibited.Green synthesis of nanoparticles is regarded as a safe and non-toxic process over conventional synthesis. Owing to the medicinal value of biologically derived biomolecules and utilizing them in synergy with nanoscience to offer more accurate therapeutic options to various diseases is an emerging field. One such study we present here with highlights of the synthesis and efficacy of biogenic silver nanoparticles produced from the extract of  Cancer is a life-threatening disease that is responsible for the majority of fatalities worldwide . ConventVerticillium sp, Fusarium oxysporum, Penicillium fungi, Trichoderma reesei, and Aspergillus fumigates. Ag+ ions are trapped at the cell surface after electrostatically interacting with negatively charged carboxylate groups in the mycelial cell wall  for Ag-NPs synthesis and found that it exhibited antibacterial, anticancer, and wound healing properties. These Ag-NPs are prospective drug leads due to the positive outcomes of prior research investigations as well as their low cost. It becomes indispensable to design a biocompatible and prolific nanoparticle that can specifically target HeLa carcinoma cells for therapeutic efficacy with reduced side effects on normal cells.In order to improve biological applications of Ag-NPs as therapeutic agents, the advancement of environmentally sustainable technology in material synthesis is of significant importance. To overcome their potential hazard and toxic effect, green synthesis have shown exceptional recognition and is preferred over chemical and physical methods because it is cost effective, less toxic, eco-friendly, requires less energy, gives high productivity and significantly biocompatible with high reduction potential . These bell wall , followiell wall . The funell wall  in orderell wall  and promell wall . The nan-7 cells . Hu et aAspergillus niger SAP2211 (a new strain) isolated from a marine sponge, as well as their subsequent characterization to determine standard nano-quality. The synthesized Ag-NPs was tested for antibacterial activity against pathogenic bacteria such as, Staphylococcus aureus, Escherichia coli, and Salmonella typhi. The anti-cancerous properties were examined in vitro in HeLa carcinoma cells and assessed cell viability by MTT assay, apoptosis by DAPI and annexin V FITC/PI, cell migration by wound healing scratch assay, and invasiveness assessed by transwell matrigel invasion assay. The anti-angiogenic potentiality were assessed in vivo utilizing the chorioallantoic membrane (CAM) model with commercial doxorubicin serving as positive control. The resulting Ag-NPs showed effective antimicrobial, anti-cancerous and anti-angiogenic potential against cervical HeLa cells with no adverse effect on normal cell, providing a platform for the use of this biogenic nano silver particles as potent antimicrobial, anti-cancerous and an anti-angiogenic agents.The current research focuses on the production of silver nanoparticles from All the chemicals and reagents used in the investigation are of molecular grade and purchased from HiMEDIA, Takara, and Sigma-Aldrich laboratories.Marine sponges were collected from intertidal and subtidal regions  of Marina Beach sea coast Chennai, India. The sponges were air dried for a period of 3–5 days and were crushed into powder.−1–10–7. A volume of 0.1 ml of each dilution was inoculated aseptically in 2 ml of Potato Dextrose Agar (PDA) plates and incubated at 37°C for 72 h. The fungal isolates were subculture on PDA plates in order to obtain pure culture. Pure isolates were then maintained in the laboratory at 4°C in a refrigerator for further studies.One gram of crushed sponge was serially diluted in sterilized distilled water to a concentration of 1020i) for its color, shape, chain morphology, hyphae and mycelium structure.The colony morphology of fungal isolates was characterized by Lactophenol Cotton Blue Mounting and was observed under Olympus trinocular light microscope (CHR DNA mini kit (Qiagen). Both quantity and quality of DNA were analyzed in 1% Agarose Tris Acetate EDTA gel. Forward primer-ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and reverse primers-ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) . The amplicon 18s rDNA gene was sequenced using an automated ABI–DNA sequencer (Applied Biosystems 3500) at Centre for Plant Molecular Biology, Osmania University. BLAST was carried out with the NCBI Genbank database using the fungal 18s rDNA ITS gene sequence and deposited in Genbank/EMBL. Sequences were identified and matched based on maximum identity values using the multiple alignment tool Clustal W. MEGA10 was used to generate the phylogenetic tree.   were useic tree. .Aspergillus niger was cultured in Potato Dextrose Broth (PDB) in a flask then incubated at 25°C at pH 6.0 for 3 days in a rotary orbital shaker at a speed of 200 rpm. Following incubation, the biomass was separated using Whatmann No.1 filter paper and was aseptically washed with sterile distilled water to remove the remaining medium components. Biomass (20 g) was mixed with 200 ml of distilled water in a 500 ml Erlenmeyer flask and incubated  in a rotary shaker. The cell suspension after incubation was filtered with Whatmann No.1 filter paper and collected for nanoparticles synthesis. Equal quantities (1:1 ratio) of both filtered supernatant and AgNO3 (1 mM) were mixed in 250 ml Erlenmeyer flask and kept in a shaker  and were confirmed by change in its color.Ag-NPs was synthesized following the methodology adopted by The prepared Ag-NPs were characterized to determine their size, morphology and crystallinity by using UV-visible spectroscopy (Shimadzu UV-1800), Fourier transform infra-red spectrophotometer (Jasco FT/IR-6300), X-ray diffractometer (Shimadzu XRD-7000), Scanning electron microscope  and Transmission electron microscope .The reduced silver ions were analyzed in the range of 300–600 nm by UV-vis. The size and morphology of the synthesized nanoparticles was determined by Scanning electron microscopy. The sample was filtered through Millipore filters of 0.2 µ to remove any contaminants. Later loaded onto a stub and coated with platinum and were intended mainly to prepare specimens for SEM observation. The size and shape of Ag-NPs were determined by TEM for which the Ag-NPs sample was diluted upto 100 times and were dropped dried on a carbon-coated Cu grid  for analysis. The size distributions of the prepared Ag-NPs on the acquired TEM images were assessed using Originpro.α radiation with λ = 1.5406 Å and 2θ (Bragg angle) ranging between 10° and 80° in steps of 0.02° with sampling time of 0.60 s per step. Debye Scherrer equation was employed to calculate the crystallite size .XRD patterns of dried Ag-NPs was analyzed by Philips X-Ray diffractometer  using Cu-K−1 in infrared spectrum.The pellet was made from dried Ag-NPs with potassium bromide (IR grade) in 1:100 ratio for FTIR analysis. Diffused reflectance was recorded in the range of 4,000–400 cmStaphylococcus aureus (MTCC 96), Escherichia coli (MTCC 443) and Salmonella typhi (MTCC 98) were procured from Department of Microbiology, Osmania University and maintained in Luria-Bertani (LB) broth at 37°C for 24 h in orbital shaker incubator.The bacterial-strains The antibacterial activity of silver nanoparticles was examined using agar well diffusion technique. The broth (0.1 ml) of each strain was uniformly plated on Nutrient agar medium using sterile cotton swabs – HiMEDIA. A well (diameter: 6–8 mm) was punched aseptically using a sterile cork borer on nutrient agar plate and approximately 10 μl volume of the synthesized Ag-NPs (Stock concentration of 1 mg/ml and 500 µg/ml) was injected into the well followed by incubation. This test was carried out in triplicate. As a control, conventional antibiotics  were employed, and the zone of inhibition was determined.°C in a 5% CO2 incubator for 24 h. The cultured cells were passaged using trypsin–EDTA (0.25%) followed by centrifugation  and resuspended in 1 ml DMEM media. About 100 µl of both cell lines (7500 cells) were added to each well of 96 welled plate and incubated overnight (37°C in 5% CO2 incubator).Myocardial (H9C2) and human cervical carcinoma (HeLa) cell lines were procured from National Centre for Cell Science (NCCS), Pune and were maintained in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin antibiotics. The cells were incubated at 37°C in 5% CO2 incubator for 21 h. Untreated cells in DMEM medium were taken as negative control.Stock solution of the synthesized Ag-NPs (1 mg/ml) and doxorubicin (1 mg/ml) was filtered using 0.45 µ syringe filter and diluted with culture media to obtain 1:1 to 1:64 dilution. The defined concentration of Ag-NPs testing agent and doxorubicin (positive control) were added in respective wells and incubated at 3750 was calculated using Graph-pad Prism software.Cell viability due to biogenic Ag-NPs and doxorubicin against cultured HeLa cell lines was performed by MTT -2, 5-diphenyl tetrazolium bromide) assay and effect of same doses of Ag-NPs were tested for its toxicity on normal H9C2 cells. The degradation of cellular mitochondria has been evaluated by an estimation of blue formazan crystals produced by the reduction of MTT by means of mitochondrial succinic dehydrogenase secreted by viable cells. MTT (20 µl/well of 5 mg/ml in PBS) was added to overnight incubated 96 welled plate followed by incubation for 3–4 h. Mitochondrial dehydrogenase reduces the yellowish water-soluble MTT to water-insoluble formazan crystals that was solubilized by adding 100 µl of DMSO in each well of the plate followed by incubation of 30 min in a shaker incubator. The quantity of formazan which is directly proportional to the number of viable cells, was measured at 590 nm using a spectrophotometer (Bio-Rad). IC5 cells/well) were cultured in 6-well plates, incubated at 37°C for overnight. Considering IC50 of Ag-NPs assessed by MTT assay, the applied doses of Ag-NPs treatments  for 24 h were compared to the untreated negative controls cells and 10 µg/ml doxorubicin treated positive controls cells. All the cells were fixed with 3.8% paraformaldehyde and stained with DAPI (0.5 µg/ml in PBS) for 15 min at 37°C in dark. The cells were washed twice with PBS. The images were analyzed and captured using an inverted fluorescent microscope  under 20X magnification.The apparently induced apoptosis by the biogenic Ag-NPs was assessed by monitoring the nuclear cell morphology exposed to various concentrations of Ag-NPs using DAPI staining method. The HeLa cells (2×105 cells/well) and cultured at 37°C, 5% CO2 for 24 h. Untreated cells were considered as negative control and the wells containing 80% confluence (approximately) were taken for the scratch test. An even scratch on cell monolayer was made with a sterile p10 tip micropipette and care was taken to avoid any possible variation in scratch width in treated and control cells, and the floating cells were washed with PBS to clean the edges. The cells were incubated with DMEM complete medium and treated with 5, 10 and 15 μg/ml of Ag-NPs and 10 µg/ml of doxorubicin. Immediately after the procedure i.e. at 0 h the region of initial migration was measured and subsequent measurements were taken after 24 and 40 h of incubations at 37°C, under inverted fluorescence microscope , and analyzed with ImageJ software  and statistically analyzed. The percentage of relative migration of NP-treated and untreated cells was determined based on the following equation:Scratch wound healing assay is convenient and widely performed to determine cell migration capabilities of individual cells, it measures the expansion of individual cell number on edge surface of the scratch . In the 5 cells per well, and the lower chamber was filled with DMEM medium (700 μl) supplemented with 12% FBS. The cells were treated with 10 and 15 μg/ml of Ag-NPs for 18 h at 37°C and after incubation; the cells were washed twice with PBS. The cells were then fixed with 3.7% formaldehyde for 2 min at room temperature followed by washing with PBS. The cells were permeabilized with 100% methanol for 20 min at room temperature and washed twice with PBS and were stained with crystal violet for 15 min at room temperature followed by washing twice with PBS. Remaining non-invasive cells from both treated and control were removed using cotton swabs. The inhibition of cell invasion ability was assessed by counting the cells and the images were captured at 40X under an inverted microscope .The invasion of Ag-NPs treated HeLa and control (untreated HeLa) cells was performed using transwell chambers . The chamber was pre-coated with Matrigel , and later HeLa cells were seeded into the upper chamber with serum free media at a density of 2 × 106 cells/dish) were incubated at 37°C for 24 h, and further treated with various doses of Ag-NPs (5 and 10 μg/ml) then re-incubated for another 24 h. The control cells were not treated with NPs. The cells were trypsinized and the floating cells were pelleted by centrifugation at 2,500 rpm for 2 min. The obtained pellet was washed with ice-cold PBS (1X) and resuspended in 100 μl binding buffer (1X). The cells were stained with both Annexin V-FITC (5 μl) and propidium iodide (10 μl) in dark for 15 min at room temperature and were diluted with 400 μl of binding buffer. Apoptotic and necrotic cells were evaluated by flow cytometer  and data analysis was performed using FACs Cell Quest Pro Software.Apoptosis induced by the Ag-NPs was assessed using an Annexin V-FITC apoptosis detection kit . HeLa cells , eggs treated with HeLa cells taken as positive control, eggs treated with 5, 10, and 15 µg/ml concentrations of Ag-NPs but with no HeLa cells, eggs treated with HeLa cells along with 10 µg/ml doxorubicin and eggs treated with both the HeLa cells as well as all the above doses of Ag-NPs.The surface of the eggs was cleaned with disinfectant prior to the treatment, egg candler was used to monitor the blood vessels and, a small hole was made and opened on snub side using dissection needle in the biosafety hood. On 11th day of incubation, a sterile 33G needle was used to inject 100 µl of individual doses of biogenic Ag-NPs  to both HeLa cell treated CAM and normal CAM surface, and with doxorubicin (10 µg/ml) as positive control. The hole(s) were covered with sterile wax and incubated at 37°C in a humidified incubator for 72 h, all the experiments were setup in triplicates. The eggs were examined for vascularization on the 14th day of incubation using stereomicroscope . The number of blood vessels in all the eggs was evaluated in percent values.p < 0.05) followed by Tukey’s test.The statistical analysis was performed using Graphpad Prism version 6 software, USA. The outcomes of three separate experiments were interpreted as mean ± S.D. and data analysis was performed using both one-way and two-way analysis of variance  groups of the primary amide due to proteins of A. niger and 2,912.46 cm−1 corresponds to O–H stretching of carboxylic group of protein. The reports documented that amino acid residues and peptide carbonyl groups have a strong affinity to bind with metals and serve as an encapsulating agent, thus preventing agglomeration of nanoparticles  which are spherical and oval in shape showing aggregation ofAg- NPs . TEM desSalmonella typhi, Escherichia coli) and gram positive (Staphylococcus aureus) pathogenic bacteria by agar well diffusion method, and compared with standard antibiotics like tetracycline and ciprofloxacin (10 µg/well). The Ag-NPs were found to be effective at all concentrations for the studied bacteria  was investigated against both gram negative  of early apoptotic population in Ag-NPs treatments, with control demonstrating 6.86%  PI (−)) and lower right demonstrates early apoptotic stage (Annexin (+) PI (−)), upper left quadrant shows necrotic cells (Annexin (−) PI (+)) and upper right (Annexin (+) PI (+)) gives count for late apoptotic phase. Results revealed that Ag-NPs treatment on HeLa cells for 24 h with 5 and 10 µg/ml Ag-NPs induced apoptosis in a concentration dependent manner . Enhanceng 6.86% . It is cng 6.86%  in 5 µg/ng 6.86%  in 10 µgng 6.86% . It is ein vivo with inhibitors and stimulators. The negative control in this experiment was eggs that had not been exposed to HeLa cells, and the vascular development in the negative control was assumed to be 100% for the measurement of angiogenesis percentage. Comparison of the mean of the number of blood vessel and percentage of angiogenesis are shown in p < 0.05) of HeLa cells inoculated CAM cells. The CAM arrangement tends to be clustered with obtruded blood vessel formation or destroyed vascular organization in all the effective dosages of nanoparticles in comparison to control groups. According to our findings, these silver nanoparticles have a dose-dependent cytotoxic impact on blood vessel endothelial cells, limiting blood vessel development in CAM.The CAM assay is a dependable method to study angiogenesis A. niger isolated from marine sponge are well documented for natural products like asperic acid, asperazine, hexylitaconic acid, and malformin C, 3,3′-bicoumarin bicoumanigrin, 6-pyridinone derivates (aspernigrins A and B), furan and pyrano pyrroles pyranonigrins  of the synthesized Ag-NPs . The UV–+ ions to enter the cell and lipopolysaccharide layer comprising of negative charge which attracts Ag+ ions causing enhanced uptake leading to destruction of cell wall  usually binds to the receptor of endothelial cells by activating the PI3K/Akt pathway and thereby lead to angiogenesis . GurunatA. niger inhabiting marine sponge. The biogenic Ag-NPs was highly stable with size lesser than 50 nm and crystalline nature. In the biological use of Ag-NPs, we discovered that the Ag-NPs exhibits promising antimicrobial efficacy against the studied pathogenic bacteria. Meanwhile, in vitro and in vivo assessment have demonstrated concentration dependent anti-proliferative, anti-invasive, pro-apoptotic and anti-angiogenic activities against HeLa cell lines. The results provides an insight on the use of biogenic Ag-NPs for future therapeutic application, as an alternative to commercial available drugs, with which further studies needs to be carried out.The present investigation concerns with novel biosynthesized Ag-NPs using