The purpose of this study was to evaluate the safety and efficacy of single-site laparoscopic extraperitoneal hernia sac ligation with an epidural needle for incarcerated ovarian hernias in infants and young children.The clinical data of 38 infants with incarcerated ovarian hernias who underwent single-site laparoscopic extradural needle extraperitoneal hernia sac ligation from January 2015 to January 2018 were retrospectively analysed.All procedures were successfully performed using laparoscopy with no need for conversion to open surgery. The time of hospital stay was 1.30 ± 0.39 days. During hospitalization and follow-up, there were no complications, such as intestinal or bladder injury, abdominal wall vascular injury, ovarian atrophy, hernia recurrence or contralateral indirect hernia. However, three patients experienced complications, including two cases of poor healing of the umbilical incision and one case of suture granuloma.Single-site laparoscopic high ligation of the extraperitoneal hernia sac with an epidural needle is a safe and feasible method for the treatment of incarcerated ovarian hernias in infants and young children. It has the advantages of minimal trauma, no scarring and good cosmetic effects. An incarcerated inguinal hernia is formed by abdominal organs that cannot be returned after entering the hernia sac and remain in the hernia sac. It is the most common complication of inguinal hernia in children. If it is not handled in time, it can lead to ischaemia and necrosis of the hernia contents, resulting in serious consequences. The characteristics of embryonic development and inguinal anatomy in female children are different from those in male children. A small evagination of the parietal peritoneum, called the canal of Nuck, accompanies the round ligament through the inguinal canal to the labium majorum . The canThis study was approved by the ethics committee of Fujian Maternity and Child Health Hospital, Affiliated Hospital of Fujian Medical University, and strictly adhered to the tenets of the Declaration of Helsinki. All patients’ guardians signed an informed consent form before the operation.Patients met the inclusion criteria if they presented with incarcerated ovarian hernia. Patients were excluded from this study for the following reasons: (1) their general condition was poor, with a history of peritonitis and systemic poisoning symptoms or serious medical diseases such as cardiopulmonary dysfunction; (2) they had a history of abdominal surgery; (3) they had a recurrent inguinal hernia; (4) their incarcerated ovaries were necrotic and needed to be replaced by ovariectomy; or (5) they refused to consent to the operation or cooperate during the follow-up schedule.A total of 46 patients with incarcerated ovarian hernia in our hospital from January 2015 to January 2018 were included in this study. Among them, 8 patients were excluded from the study for the following reasons: 4 with a poor general condition, 1 with a history of abdominal surgery, 1 with a recurrent inguinal hernia and 2 with necrotic ovaries. Finally, this study retrospectively analysed the clinical data of 38 patients, including preoperative general data and intraoperative, postoperative and follow-up data. All patients were diagnosed by physical examination, combined with clinical manifestations and inguinal colour Doppler ultrasonography, and manual reduction failed in all cases. There were a total of 13 patients with left hernia and 25 patients with right hernia, and the average age and weight of the patients were 5 months (21 days to 11 months) and 5.6 kg (2.7–11.1 kg), respectively . We then explored the abdominal cavity: a small amount of clear ascites was found in the abdominal cavity, the ring of the affected inguinal canal was not closed, and the oviduct continued to the inguinal canal with slight oedema and hyperaemia  successfully underwent laparoscopic high ligation of the extraperitoneal hernia sac with an epidural needle under single-site laparoscopy. In this group, seven cases were incarcerated for a long period of time, and the suspected disorder of incarcerated ovarian blood supply was found during the operation. After laparoscopic-assisted reduction, the observation time was prolonged, and the blood supply to the ovaries recovered. The operative times of the unilateral and bilateral ovarian hernias were 32 (26–41) min and 45 (40–56) min, respectively. The time of hospital stay was 1.30 ± 0.39 days (Table Incarcerated ovarian hernia is a common type of incarcerated inguinal hernia and one of the emergency diseases in paediatric surgery. As recently reported , 8, the For female infants with incarcerated ovarian hernias, emergency surgical treatment is needed when manual reduction fails. The traditional surgical approach is to perform high ligation of the hernia sac through the inguinal incision. For children with incarcerated hernias, local inguinal tissue congestion and oedema are obvious, and the operation is difficult. To reduce the difficulty of the operation and tissue identification, the surgical incision is often larger than that of non-incarcerated hernia, the structure of the inguinal canal is more traumatic, and the scar is more obvious after operation. Family members of female children often have higher requirements for postoperative cosmetology. Meanwhile, traditional operations cannot detect and properly address occult contralateral inguinal hernias, while bilateral and occult contralateral inguinal hernias are more common in infants and young children’s inguinal hernias , 10. ZhoLaparoscopic high ligation of the extraperitoneal hernia sac with an epidural needle is a simple, reliable and simple puncture technique that can be accomplished by external ligation. It has the advantages of minimal trauma, fast recovery, low recurrence rate and good cosmetic effects –24. CompAlthough this retrospective study had a certain size, there are still several limitations. First, this was a retrospective study with a limited number of patients from a single centre, and more research from multiple centres is needed to assess the effectiveness and complications of this technique. Second, the median follow-up duration was relatively short, and a longer follow-up period is warranted.In conclusion, single-site laparoscopic high ligation of the extraperitoneal hernia sac with an epidural needle for ovarian incarcerated hernias in infants is a safe and feasible alternative to conventional surgery. The cosmetic results were impressive, leading to less physical and psychological trauma, and the follow-up results were promising.


The hollow nanofibers also outperformed the other catalysts in terms of photocatalytic degradation of MB dye, at 85.5%. The optimum catalyst loading, dye concentration, pH, and H2O2 concentration were determined at 0.75 g/L, 10 ppm, pH 11, and 10 mM, respectively. The highest degradation of methylene blue dye achieved was 95.2% after 4 h of UV irradiation.Environmental crisis and water contamination have led to worldwide exploration for advanced technologies for wastewater treatment, and one of them is photocatalytic degradation. A one-dimensional hollow nanofiber with enhanced photocatalytic properties is considered a promising material to be applied in the field. Therefore, we synthesized titanium dioxide hollow nanofibers (THNF) with extended surface area, light-harvesting properties and an anatase–rutile heterojunction via a template synthesis method and followed by a calcination process. The effect of calcination temperature on the formation and properties of THNF were determined and the possible mechanism of THNF formation was proposed. THNF nanofibers produced at 600 °C consisted of a mixture of 24.2% anatase and 75.8% rutile, with a specific surface area of 81.2776 m Water contamination caused by domestic wastes, industrial chemicals, fertilizers, and organic dyes has become one of the global environmental concerns . Organic2) is one of the most commonly used metal oxides owing to its high stability, low cost, abundance, stability, efficient photoactivity, and safety to human beings and the environment 4 was used as the precursor. A hydrolysis reaction occurs when the Ti-OR group is substituted with water to Ti-OH moieties, as presented in Equation (4). Then, the condensation process forms Ti-O-Ti or Ti-OH-Ti oxide networks, as shown in Equation (5). Nucleation and growth of TiO2 particles occur when the oxide network undergoes further condensation, as shown in Equation (6). Since the TiO2 sol-gel has been successfully coated on the PAN nanofibers, the obtained TiO2 nanoparticles impregnate the surface of PAN nanofibers, forming a TPNF nanocomposite. The overall reaction is shown in Equation (7). The mechanism of TiO2 nanoparticle growth on the PAN surface is illustrated in Generally, TiOHydrolysis of TTIP:4:Condensation of Ti(OH)Further condensation:Overall reaction:2 hollow nanofibers have been successfully synthesized via template synthesis. The obtained hollow nanofibers exhibit admirable photocatalytic performance attributed to the anatase–rutile heterojunction creation, extended surface area for UV and dye molecules adsorption, and light-harvesting effect in the hollow cavities of the nanofibers. The calcination temperature plays an important role in the formation of the hollow structures and in determining the physicochemical properties of the TiO2 hollow nanofibers. The TiO2 hollow nanofibers were successfully synthesized at 600 °C and THNF600 has the best characteristics in terms of morphology, surface area, crystallinity, and optical properties to be used for the photodegradation process. We had also investigated the effects of operating parameters on the photodegradation of MB dye solution. The highest photocatalytic degradation efficiency was obtained at catalyst loading = 0.75 g/L, MB dye concentration = 10 ppm, pH = 11 and H2O2 concentration = 10 mM. Lastly, the mechanism of TiO2 hollow nanofiber formation was also proposed.In this work, TiO


Data on comprehensive population-based surveillance of antimicrobial resistance is lacking. In low- and middle-income countries, the challenges are high due to weak laboratory capacity, poor health systems governance, lack of health information systems, and limited resources. Developing countries struggle with political and social dilemma, and bear a high health and economic burden of communicable diseases. Available data are fragmented and lack representativeness which limits their use to advice health policy makers and orientate the efficient allocation of funding and financial resources on programs to mitigate resistance. Low-quality data means soaring rates of antimicrobial resistance and the inability to track and map the spread of resistance, detect early outbreaks, and set national health policy to tackle resistance. Here, we review the barriers and limitations of conducting effective antimicrobial resistance surveillance, and we highlight multiple incremental approaches that may offer opportunities to strengthen population-based surveillance if tailored to the context of each country. Low- and Middle-income countries bear the highest burdens of communicable diseases with potentially the least resources, and limited data on the epidemiology and burden of antimicrobial resistance (AMR) , 2. Currwww.databank.worldbank.org › data › site-content › CLASS). According to the World Health Organization (2019) report [The World Bank list of economies June 2020) classifies 29 countries as low-income and 106 countries as middle-income  report , global ) report . Donor f) report . Poorly ) report . These f) report , 16. Gov) report . In LICs) report . Communi) report , 18. Vec) report . In the zation 20 report [20 classi) report . In LMIC) report , 23. Hea) report , 23. TheIn LMICs, the basic requirements for a functional laboratory infrastructure are not met –26. ThesUnderstaffing –26, 29, Poor communication between laboratory staff and the medical team  is cruciOmelet and colleagues (2018)  discusseLaboratory Guidance for the selection, sampling, and transport of specimens is absent . There iExternal funding to strengthen the laboratory capacity and implement AMR surveillance programs is granted by agencies like the Fleming fund, the WHO, and U.S Centers for Disease prevention and Control (CDC) . FundingOne of the five strategic goals of the WHO global action plan is to strengthen data on AMR through surveillance and research . Data caData management is one of the main challenges of AMR surveillance –53. The In 2015, WHO launched the GLASS that establishes a standardized approach for the collection, analyses, and data sharing on AMR worldwide . The GLAHaemophilus influenza [Pharmaceutical companies establish global networks examining bacterial susceptibility mainly to evaluate drug performance , 14. Thenfluenza , 60. TheAn academic network may offer high-quality data and has many advantages compared with a pharma network , 14. ThePrivate laboratories may play a major role in the provision of high-quality data on AMR surveillance if they are accredited and operate according to quality standards provision of services compared with public laboratories . In SoutThe countries included in the study provide a snapshot of data on AMR surveillance programs and the epidemiology of AMR in LMICs located in different continents Table .Bangladesh is a lower middle-income country (LMIC) with a population of 163.05 Million. Bangladesh is enrolled in the GLASS and has an established national coordinating center with an in-place national action plan [mcr) genes were found in Bangladeshi veterinary and environmental sources [Escherichia coli [Salmonella Typhi [Mycobacterium tuberculosis [Vibrio cholerae [Proteus mirabilis, Streptococcus pneumoniae, Haemophilus influenza [Acinetobacter baumanni (A. baumannii) [Pseudomonas aeruginosa (P. aeruginosa) [Staphylococcus aureus [Klebsiella pneumoniae (K. pneumoniae) [Clostridium difficile [ion plan , 66. Theion plan . AMR preion plan –70. The  sources –74. Anti sources –78, gast sources , 80, uri sources , 82, res sources , skin an sources , blood-b sources . Most sthia coli , 85, Salla Typhi , Mycobacrculosis , 77, Vibcholerae , Proteusnfluenza , 87, Aciumannii) , Pseudoms aureus , Klebsieumoniae) , ClostriBrazil is an Upper Middle-Income country (UMIC) with a population of 211.05 Million. Brazil is enrolled in the GLASS and has an established national coordinating center with an in-place action plan [among more than 20,000 genes detected, the blaKPC gene isolated from K. pneumoniae predominates, followed by blaOXA-23 Acinetobacter spp. The blaOXA-48, known to be highly prevalent in European countries is rarely found in Brazil. According to GLASS 2020 early implementation results, the prevalence of S. pneumoniae, S.aureus, Escherichia coli (E. coli), Acinetobacter spp. and Salmonella spp. are between 70 to100% reported in blood culture according to AST results [. The rates of MRSA are high estimated up to 60% and are related to an endemic Brazilian clone. Resistance to vancomycin was first attributed to Enterococcus faecalis, which differs from the reported epidemiology of Enterococci in Europe and America [K. pneumoniae and Escherichia coli (E.coli) isolates producing ESBL have a much higher prevalence . Other Gram-negative bacteria (GNB) such as carbapenem-resistant K. pneumoniae and carbapenem-resistant non-fermenting gram-negative bacilli (NFGNB) are frequently reported in different studies conducted in various states in Brazil [P.aeruginosa and OXA-23 A. baumannii complex enzymes where a phenotype has also emerged in these isolates that are only susceptible to Colistin [ion plan . The Minion plan . By 2022ion plan  expects  results , 90. The America . K. pneun Brazil , 91–93 AIndia is an LMIC with a population of 3.37 billion. India is enrolled in the GLASS and has a national coordinating center, and established a national action plan [E. coli [Salmonella species (spp.) [Shigella spp.[Pseudomonas spp. [Acinetobacter spp. [K. pneumonia and almost half of all P. aeruginosa found resistant to fluoroquinolones and third-generation cephalosporins [. In 2019, under the National Antimicrobial Resistance surveillance network (NARS-Net India) the National Centre for Disease Control received AMR Surveillance data from 21 sentinel surveillance laboratories in different States. E. coli was the most prevalent pathogen (33%) in inpatients and outpatient settings. The second commonly detected pathogens were Klebsiella spp. (22%) isolated in ICU where 5% of total blood isolates were resistant to colistin. Klebsiella spp. and E. coli showed high resistance to carbapenem and 3rd and 4th generation cephalosporins. S. aureus constitute (18%) of the total reported isolates with 66% resistance to cefoxitin and 1% resistance to linezolid. Other gram-positive bacteria include Enterococcus spp. with 5% resistance to linezolid and 13% resistance to vancomycin. Other isolated pathogens include Pseudomonas spp. (10%), Enterococcus spp. (9%), Acinetobacter spp. (8%) and Salmonella Typhi and Paratyphi (< 1%). High rates of resistant pathogens to most antibiotics in ICU settings is a major concern in India [ion plan , 66. In ion plan  and inition plan , 96. In ion plan  to promoion plan . AMR in ion plan . Researcion plan –107. In [E. coli , 109, Sas (spp.) , 111, Shella spp., 113, Psnas spp. , 109, anter spp. , 107. A osporins . Resistaosporins . In 2019in India , 116.Lebanon is a UMIC with a population of 6.86 million. Lebanon is enrolled in the GLASS and has an established national coordinating center and a national action plan on AMR [Enterobacteriaceae [E. coli and K. pneumoniae [A.baumanii[-mediated infections [. Very few studies tackled the alarming spread of gram-positive resistant bacteria [Methicillin-Resistant Staphylococcus Aureus (MRSA) [Streptococcus spp. [n on AMR , 66.Publn on AMR , 117–125n on AMR , 123–125eriaceae , 127. Reeumoniae , 128–131.baumanii OXA-48 [fections . Very febacteria , 125, Mes (MRSA) , and Strcus spp. , 134. DaMalaysia is a UMIC with a population of 31.95 million. Malaysia is enrolled in the GLASS and has an established national coordinating center with an in-place national action plan [ion plan , 66.Staphylococcus aureus, Streptococcus pneumonia (S.pneumoniae), E.coli, K.pneumoniae, A.baumannii, P. aeruginosa, and enterococci. Data from the NSAR program showed a high burden of extended-spectrum B-lactamase-(ESBL) producing Enterobacteriaceae and is of concern in the hospitals [resistant K. pneumoniae (CRKP) in 2010 showed increased spread from 0.3% in 2011 to 3.5% in 2018. Colistin-resistant bacteria showed a widespread trend among hospitalized patients in recent years [In Malaysia, a study in the early 1990s has reported the resistance patterns of more than 36,000 microorganisms isolated in 6 general hospitals . The Natospitals . The hignt years , 137.Staphylococcus aureus (S. aureus), S. pneumonia, and K. pneumoniae are decreasing. For example, the MRSA rate decreased from 19.4% in 2018 to 15.0% in 2019. Resistance to vancomycin was lower in 2019 in both Enterococcus faecalis, Enterococcus faecium, and polymyxin B resistance remained at a low level. On the contrary, resistance rates have increased from the previous year in the majority of antimicrobials tested for A.baumannii and P. aeruginosa, including non-susceptibility to carbapenems (imipenem and meropenem). A. baumannii is isolated from patients in various departments [Escherichia coli isolated from urine and showed resistance rate to cefepime, cefuroxime, and ciprofloxacin [In 2019, NSAR  reportedartments . Ampicilfloxacin , 139.South Africa is a UMIC with a population of 58.56 million. South Africa is enrolled in the GLASS and has an established national coordinating center with an in-place national action plan [ion plan , 66.Enterococcus faecium (E. faecium), S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp.) had varied resistance rates and patterns across the country, comprising between 24 and 33% of all cultures. It was surprising that 75% of antimicrobial use in South Africa was in humans, rather than the higher rates of use in animals from other countries [K. pneumoniae had 60–70% resistance patterns with ESBL, limiting the use of cephalosporins as first-line therapy, while there is an emerging carbapenem resistance, albeit lower. E. coli ESBL showed 25% resistance patterns and a worrying increased resistance to quinolones . P. aeruginosa and multi-drug resistant A. baumanni were only susceptible to colistin [MRSA was found [K. pneumoniae had 60–70% resistance patterns with ESBL, limiting the use of cephalosporins as first-line therapy, while there is an emerging carbapenem resistance, albeit lower. Interestingly a decline in MRSA was found [S. aureus, E. Coli, K. pneumoniae, and P. aeruginosa, with AMR trending upwards over time from between 30–60% in the early period and up to 64–81% in the later period [Staphylococcus spp. and up to 30% ESBL-producing K. pneumoniae[The national first report of the five years 2012–2017  showed tountries . K. pneucolistin . Interesas found . There aas found . These ras found . Recent as found . Numerour period . Interesr period . The chaneumoniae. While mneumoniae, 145.E. coli (19% in the public sector versus 36% in the private sector), A. baumannii (14% public versus 4% private), P. aeruginosa (7% public versus 11% private) and S. aureus (27% public versus 17% private), however concerning was the rapidly decreasing carbapenem susceptibility among Enterobacteriaceae [K. pneumoniae. Similar results were found in a longer period using data from KwaZulu-Natal [A. baumanii specimens examined, with only 5.4% resistant to this medication [A. baumanii is practiced [K. pneumoniae soon as this defeats the carbapenem group of antimicrobials [Other studies have monitored the prevalence of various mutations and resistance patterns in parts of SA and show that there is a difference in the patterns between the private and public sectors, specifically eriaceae . Susceptlu-Natal , who alsdication . These lracticed , 150. Wocrobials , 152.Ukraine is an LMIC with a population of 41.98 million. Ukraine is enrolled in the CAESAR network [S. pneumonia, and H. influenzae isolates from community-acquired respiratory tract infections showed high susceptibility to tested antibiotics and an increase in antibiotic resistance to trimethoprim/sulfamethoxazole and macrolides among S. pneumoniae[the EUCAST/CLSI and pharmacokinetics and pharmacodynamics breakpoints165. Another study conducted in hospital settings to examine the pathogens associated with surgical site infections showed that most isolated pathogens were Gram-positive bacteria where staphylococci showed the highest resistance to Gentamycin and ceftibuten. Besides, data showed the prevalence of 48.1% MRSA and 36.6% Methicillin-resistant S. epidermidis (MRSE) while vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant S. epidermidis (VRSE) ranged from 9.3% and 18.3%154. Another study conducted in acute care settings of isolates demonstrated the high prevalence of resistant pathogens causing healthcare-associated infections (HAI). Results showed that a total of 14.2% of enterococci were resistant to vancomycin and 28.2% of isolated S. aureus were methicillin-resistant, and 35.1% of Enterobacteriaceae were resistant to third-generation cephalosporins among which the attributable highest rates of resistance to the K. pneumoniae (53.8%) and E. coli (32.1%)[P. aeruginosa and A. baumanii [E. coli [Methicillin-resistant Staphylococcus epidermidis (MRSE) and ESBL production among Enterobacteriaceae in postpartum mastitis [ network , 66. In  network . Publish network –160. Theneumoniae–164. Thei (32.1%). A retro[E. coli . The recmastitis  and postmastitis . To datemastitis , 170.A comprehensive AMR surveillance data is compiled from multiple sources worldwide across different sectors including the human health, animal health, and the agricultural sector interface, in addition to local and regional sources including primary and tertiary hospitals, laboratories, clinics, primary clinical settings –173, phaIn LMICs, the challenges are high , 25, 26.The mainstay to strengthen the laboratory capacity is government engagement. Health is a political decision . The WHOInternational funding agencies, donors, and philanthropic organizations can invest in the manufacturing of adapted or alternative supplies and scholarship grants for post-graduate education, continuous education and training, and health systems research , 26. InvInternational initiatives, academia, Pharma, and specialized societies can also contribute to improving human resources knowledge through education and training , 26. InvThe key to secure high-quality performance is the national and international accreditation of laboratories that should undertake a periodic EQA embedded as a policy in the national action and national health plan , 25, 26.Appropriate data management is an additional issue . Better Bioinformatics and genomics offer a promising shift in paradigm –186. WitS. aureus species [However, the comparison of big data and analysis of genomes requires a professional approach that can link all this information. Effectively, Bioinformatics integrates knowledge from molecular biology, infectious disease specialty, and epidemiology to predict AMR. Multiple prediction categories like the Binary classification and Multiclass classification are used –191. The species , 192.This approach requires adequate resource allocation, interdisciplinary effort, funding, and teamwork to provide early diagnosis and increase the quality of care in the era of drug resistance. Low and middle-income countries such as in the Middle Eastern Arabian Peninsula are lagging in the research in WGS based research and might consider implementing these genomics techniques to gather AMR data for global surveillance . FinallyArtificial intelligence (AI) is a new paradigm to combat AMR[ombat AMR. AI modeombat AMR, controlombat AMR, 197.Theombat AMR.The phenombat AMR, 199. Thombat AMR, 199. Boombat AMR. The resombat AMR. The pheombat AMR. Besidesombat AMR.The WGS ombat AMR. On a glombat AMR. AMR surombat AMR, 200, inombat AMR, 200. Thombat AMR. AI offeombat AMR–203.The ombat AMR, 204. Thombat AMR, 205–207ombat AMR. For WGSombat AMR, 209.Theombat AMR. The SVMombat AMR. SVM is ombat AMR that is ombat AMR.AMR surveillance from a “One Health” perspective is needed in LMICs for data comparability cross-borders and for mapping and tracking the spread of resistance. Aggregated data can advise the estimation of the economic burden of AMR from societal and ecosystem perspectives and examining the cost-effectiveness of the current infection control and prevention program. Prioritizing health expenditures is crucial in LICs. Another topic of interest is working on sampling the environmental resistome in these regions to explore the resistance determinants and understand the niches that contributed or will potentially contribute to infections with antibiotic resistance microorganisms.A significant progress is achieved toward standardization of a population-based AMR surveillance in LMICs. The mainstay remains that “One size-fits-all” global action cannot be applicable, and an efficient action plan starts by an understanding of the particularities of each country and by aligning regional, national and international efforts. The government commitment to health as a national priority is the key to strengthen regulations, follow-up the implementation of a national action plan and control anthropogenic activities on antibiotic use in different sectors.


Mechanistic analyses show that DANCR can serve as miRNA sponges, stabilize mRNAs, and interact with proteins. Recent research reveals that DANCR can be detected in many body fluids such as serum, plasma, and exosomes, providing a quick and convenient method for cancer monitor. Thus DANCR can be used as a promising diagnostic and prognostic biomarker and therapeutic target for various types of cancer. This review focuses on the role and mechanism of DANCR in cancer progression with an emphasis on the clinical significance of DANCR in human cancers.Long non-coding RNA (lncRNA) DANCR —differentiation antagonizing non-protein coding RNA, was first reported in 2012 to suppress differentiation of epithelial cells. Emerging evidence demonstrates that DANCR is a cancer-associated lncRNA abnormally expressed in many cancers . Increasing studies suggest that the dysregulation of DANCR plays critical roles in cancer cell proliferation, apoptosis, migration, invasion, and chemoresistance  LncRNAs are non-coding RNAs (ncRNAs) consisting of longer than 200 nucleotides in length without initiation codon and termination codon. They were initially considered as “junk” transcriptional products without biological functions, thus attracting limited attention among scientists . HoweverLncRNAs also play essential roles in human cancers. The previous studies have indicated that many lncRNAs are dysregulated in cancers . AberranDifferentiation antagonizing non-protein coding RNA (DANCR or ANCR), located on human chromosome 4q12, was first reported in 2012 to suppress differentiation of epithelial cells, and then proved to promote the stemness features of hepatocellular carcinoma cells . SubsequThe downstream regulatory mechanisms for the biological roles of DANCR in cancers are complicated . The comIn addition to binding to miRNAs, DANCR could also interact with mRNAs or proteins. For example, DANCR was verified to interact with 3′UTR of CTNNB1 mRNA in hepatocellular carcinoma, thus competitively blocking miRNA site and reversing miRNA-mediated CTNNB1 suppression . SimilarAnother function of DANCR is epigenetic regulation. EZH2, a histone methyltransferase of polycomb repressive complex 2 (PRC2), is a common binding protein for DANCR. By interacting with EZH2, DANCR could epigenetically silence target gene transcription. In cholangiocarcinoma, DANCR-EZH2 interaction promoted FBP1 silence by regulating histone methylation of FBP1 promoter and then exacerbating tumorigenesis .6A modification  is one of the most common oral cavity malignancies, known for its aggressive biological behavior . AlthougIn vitro studies showed that DANCR could enhance TSCC cell proliferation, migration, and invasion. The molecular mechanism study suggested that DANCR act as a ceRNA to sponge miR-135a-5p and upregulate Kruppel-like Factor 8 (KLF8) expression. Similarly, knockdown of DANCR inhibited tumor growth and KLF8 expression in vivo, suggesting that DANCR may play an essential role via miR-135a-5p/KLF8 axis  is one of the most frequent cancers in the digestive system and ranks the seventh leading cause of death in cancers . Though ZNF750. The mutations or deletions of ZNF750 were significantly associated with upregulated DANCR and poor prognosis, especially tumor metastasis in ESCC patients. Their further investigations verified that DANCR could competitively bind with miR-4707-3p and serve as a ceRNA to regulate FOXC2 expression in ESCC, which provides a novel DANCR/miR-4707-3p/FOXC2 pathway regulated by ZNF750 in ESCC. In summary, DANCR is associated with ESCC progression, and it may act as a novel prognostic and therapeutic target for ESCC.Esophageal squamous cell carcinoma (ESCC) is one histological subtype of EC . Shi et Gastric cancer (GC) is a complex and heterogeneous disease, regarded as the fourth leading cause of cancer-related death . TraditiRecent studies have revealed that DANCR expression was upregulated in GC tissues  and cellChemotherapy is the primary therapy for GC patients with unresectable tumors, and cisplatin (DDP) is used as the first-line drug . HoweverColorectal cancer (CRC) is one of the most commonly diagnosed malignant neoplasms among men , 2021. Tin vivo study, the elevation of DANCR promoted tumor growth and liver metastasis of CRC. Another mechanism study by A recent study by DANCR was also overexpressed in the serum of CRC patients. Serum DANCR level was positively associated with the TNM stage. Surprisingly, serum DANCR showed a better diagnostic ability than CEA and CA199 and a better performance in distinguishing CRC and colorectal polyps. Moreover, the combination of DANCR with CEA and CA199 showed better sensitivity than the single or double combination ; Table 1Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and ranks the sixth most common neoplasm and the third leading cause of cancer-related death . SorafenDANCR was upregulated in HCC cell lines, tissues, plasma and exosomes . It was DANCR could act as sponges for multiple miRNAs including miR-216a-5p , miR-27aIn conclusion, DANCR is a crucial factor in HCC progression and may serve as a new marker for HCC diagnosis and treatment.Cholangiocarcinoma (CCA) is a rare but aggressive biliary epithelial tumor . Surgeryin vitro. miR-345-5p was predicted and verified as a target miRNA of DANCR, which regulated the expression of Twist1 (Twist-related protein 1) in CCA cells  . What isin vitro, and tumor growth in vivo  is not a common cancer type but notable for its distinctive geographical distribution pattern. Most cases occur in the east and southeast parts of Asia . NPC patin vitro. In addition, DANCR knockdown inhibited NPC tumor growth in vivo  overexpression . Wen et Lung cancer is the leading cause of cancer-related deaths worldwide, both in men and women. It is reported that nearly a quarter of cancer deaths are related to lung cancer . Non-smaUpregulated DANCR expression was detected in NSCLC tissue specimens and cell lines compared with normal counterparts. High level of DANCR was associated with larger tumor size, advanced TNM stage, and lymph node metastasis. Due to PSA testing and advances in early detection and treatment, the death rate for prostate cancer (PCa) dropped by 51% in the past two decades. However, PCa remains the second leading cause of cancer-related deaths in men, and the high rate of overdiagnosis is widely debated. Androgen deprivation therapy (ADT) is the principal treatment for advanced PCa . HoweverStrong evidence has been presented about the oncogenic roles of DANCR in PCa. DANCR was upregulated in PCa tissues and cell lines . Lu Y. eChemotherapy with Taxol  is commonly used for CRPC. However, there are obstacles to drug resistance . Ma et aOvarian cancer (OC) is typically diagnosed at an advanced stage and has no effective screening strategy . SearchiDANCR was detected upregulated in ovarian cancer tissues and cell lines . VasculaInfection of specific types of human papillomavirus (HPV) is the primary biological etiology for cervical cancers (CC). Prophylactic vaccination for HPV provides the most effective method of primary prevention against HPV-related diseases . HoweverFrom Cancer statistics 2021, breast cancer (BC) has become the most common type of malignancy among women worldwide, responsible for 15% of deaths in women . TNBC t. Though in vivo.DANCR was significantly upregulated in TNBC tissues and cell lines compared with normal ones . Sha et The molecular mechanism analysis showed that DANCR played oncogenic roles by targeting miR-216a-5p as a ceRNA in MDA-MB-231 cells (TNBC cell line) . In addiInterestingly, DANCR has also been suggested as a tumor suppressor in BC. Bladder cancer (BCa) is among the top ten most common cancer types and the second most common genitourinary malignancy globally, with approximately 550,000 new cases annually . DiagnosRenal cell carcinoma (RCC) is one of the most common cancers in the urological system, originating from the renal tubular epithelial system. Surgical resection is an available choice for patients with localized RCC. However, due to the lack of sensitivity to radiotherapy and chemotherapy in RCC, targeted therapy is necessary for individuals without conditions for surgery .Papillary thyroid cancer (PTC) is the most prevalent form of thyroid cancer with a rapidly increasing incidence without a concomitant rise in mortality . Though Glioma is the most frequent and lethal central nervous system (CNS) tumor occurring both in children and adolescents . DANCR eOsteosarcoma is one of the most common primary solid malignancies of bone and mainly occurs in adolescence. Though the cure rate for conventional treatment of osteosarcoma is close to 70%, once osteosarcoma has spread to distant organs such as lungs, the survival rates are disappointing . UnderstIncreased expression of DANCR could be detected in osteosarcoma tissues and cell lines. Enhanced DANCR was found to have a positive correlation with poor prognostic outcomes . DANCR sNot only in cancer, DANCR could also participate in various biological processes and other diseases. Many research indicated that DANCR might play important role in the differentiation of mesenchymal stem cells (MSCs). Dysregulation of lncRNAs is involved in regulating diverse malignant behaviors of cancer cells, leading to cancer progression and metastasis. It indicates that developing new diagnostic methods and therapeutic options targeting lncRNAs may be a new answer to the fight against cancers. LncRNA-DANCR, a booming researching topic in recent years, has been demonstrated to regulate many cellular functions such as proliferation, apoptosis, EMT, and CSC in various human cancers. The mechanism by which DANCR promotes tumor development is extremely complicated, including serving as a ceRNA for miRNAs, interacting with mRNAs or proteins, activating signaling pathways, and regulating epigenetic modulations. This review depicts a comprehensive picture of the biological roles of DANCR and its underlying mechanisms in cancer.In most cancers, DANCR was upregulated and acted as an oncogene. However, a minority of studies reported that DANCR functioned as a potent tumor suppressor. Even in the same cancer type, the role of DANCR contradicted with each other . This maDANCR is considered as a powerful biomarker not only in discriminating cancer patients from healthy people or patients with benign diseases but also in helping to predict the prognosis for cancer patients. Moreover, the combination of DANCR and other traditional biomarkers may enhance diagnostic efficiency. For cancer treatment, DANCR may be a promising target due to its essential role in cancers. Though DANCR was a promising biomarker and therapeutic target in cancer, more comprehensive and systematic clinical studies and more extensive sample tests are needed to further explore these complex issues. Further studies in DANCR mechanistic investigations and clinical application are still needed. Only after the mechanisms of DANCR in specific cancers have been elucidated can it likely be used for therapeutic purposes.JY, XiZ, and XF conceived the project and supervised the writing. MW and JG wrote the draft of the review. XuZ and XF provided funds and assisted with preparation of the manuscript. All authors are involved in the revision and approved the final version of the manuscript.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.


Antimicrobial resistance (AMR) in rural Latin America is not fully understood. The transmission pathways are partially known since research predominantly focuses on the urban hospital setting. The contribution to AMR from environmental factors is usually only mentioned in large-scale animal production. To understand the state of the literature on AMR in rural LA, we carried out a scoping review using the One Health (OH) perspective. OH recognises the concomitant contributions and interconnectedness of humans, animal, and the environment, thus, we used the OH perspective to select those articles adopting a holistic view of the problem. We searched original articles in English, Spanish, and Portuguese in four peer-reviewed databases and included 21 publications in the analysis. We charted data on bibliometrics, design, data collection sources, and instruments. We identified the human, animal, and environmental contributions to AMR in rural locations, and information gaps on AMR transmission routes and AMR drivers. Intensive and non-intensive animal production systems and agricultural practices were the most frequently found human contributions to AMR. Poultry, swine, cattle, and fish were the most frequent livestock mentioned as sources of AMR bacteria. Animal carriage and/or transfer of AMR determinants or bacteria was recognised as the primary contribution of livestock to the problem, while water, soil, and farming were predominant environmental contributions. We found that only 1 article out of 21 considered the OH approach as a framework for their sampling scheme, whereas 5 out 21 discussed all the three OH components. There were hardly any descriptions of humans or human waste as reservoirs for AMR in rural locations, and rural health centres or hospitals and wildlife were not represented. No studies identified mining as an anthropogenic activity driving AMR. More OH-oriented studies, with emphasis on molecular approaches—for identification and comparison of AMR genes—are sorely needed to understand better the existence of a network of interconnected transmission routes in rural Latin America and provide efficient strategies to prevent further AMR emergence. Antimicrobial resistance (AMR) is a global public health issue ,2,3, whiUsing the OH lens, we find that in humans, AMR is linked to diverse social factors such as non-adherence to regimens or doses, self-medication, and misperceptions regarding antibiotics . LikewisSpecifically, in Latin America (LA), human and veterinary use of antibiotics is loosely regulated, and antibiotics are readily available over the counter without prescription ,21,22. IThis study aims at identifying research that focuses on AMR in rural settings in Latin America (LA) using the OH lens as a framework to identify their human, animal, and environmental contributions to AMR. In addition, this work seeks to pinpoint knowledge gaps on AMR transmission routes and AMR drivers to inform researchers and raise awareness in policymakers. Specifically, this review answers the following research question: What are the concomitant contributions of humans, farm animals, and the environment on antimicrobial resistance in rural settings in LA?The steps included for our review followed the checklist outlined in the PRISMA Extension for Scoping Reviews (PRISMA-ScR) by Tricco et al.  and the The study period spanned from January 2001 until December 2018. Only original, peer-reviewed articles published in Spanish, Portuguese, and English were considered. A search strategy was designed using terminology associated with the three domains of OH and antimicrobial resistance in rural geographic environments for human populations in Latin American countries. We developed and refined key search terms with online databases prior to the article search .We excluded grey literature. Reviews and meta-analyses were excluded, but their reference lists were scanned. Articles not acknowledging or discussing all three OH components were excluded.We consulted the following electronic databases concentrating on peer-reviewed articles only: PubMed , Web of Science (multidisciplinary), Scopus (multidisciplinary), and SciELO (multidisciplinary for LA and the Caribbean). We carried out the search from 13 November to 3 December 2018. All articles were uploaded to a Mendeley database .A multi-step process was applied for the analysis of inclusion . Our iniWe defined relevant publications as any original peer-reviewed article published between 1 January 2001 to 3 December 2018; in English, Spanish, and Portuguese language that presented data from LA countries only ; referred to a rural human setting and to agricultural and/or animal-based food production activities, linked to environmental aspects, and focused on the topic of AMR. To be included, an article needed to refer to any aspects of AMR and to partially or totally match the concept of AMR as defined by the WHO: “Antimicrobial resistance (AMR) is the ability of a microorganism  to stop an antimicrobial  from working against it. As a result, standard treatments become ineffective; infections persist and may spread to others” .Considering an OH perspective, and using it as a screening tool, we searched for articles that linked to human populations in rural settings , had a connection with agricultural and/or animal production activities , and described a strong connection with AMR and the environment. All the inclusion criteria needed to be met, and thus, the criteria needed to be explicitly stated and/or discussed in the article. Finally, we excluded studies on AMR that focused only on topics that were remotely linked to agricultural and animal-based-food production activities, such as articles describing urban hospital settings or industrial food production settings; peripherally related to environmental aspects such as publications dealing with economic impacts; loosely linked to AMR discussing molecular/kinetics analyses of enzymes responsible for AMR, and describing research in parks and zoos within urban spaces, since they would not qualify as “rural”.We tabulated data extracted from the selected articles, including authors, year of publication, title, research objectives, DOI, URL, location of the study, language, and summary of the findings. We used a charting spreadsheet established a priori as a guide, which was established through team discussions when reviewing the preliminary results. If investigators from individual studies were contacted, their clarifications were included.The analysis and synthesis of literature included quantitative analysis  and qualitative analysis . For the qualitative analysis, reviewers extracted common themes that emerged from the findings, and the team discussed the results. Each article was analysed to identify the approach to study AMR and findings regarding each theme.A total of 19 articles were included in the analysis, and 2 articles were added after browsing their references .Salmonella spp. strains, and three studies were partially funded by private LA universities.The 21 studies included in the analysis originated from 8 Latin American countries: Brazil, Ecuador, Colombia, Argentina, Chile, Mexico, El Salvador, and Peru, with Brazil providing the most articles (9 of 21). All articles but one were published in 2010 and onwards, peaking with five publications in 2014. All of them were written in the English language. Nineteen studies were funded by research funders, public agencies; two did not declare their funding source. One study was partially funded by a microbiological laboratory which supplied All studies were quantitative and had a cross-sectional design except for one, which was qualitative. Two articles addressed AMR only through molecular methods, six combined phenotypic profiling and molecular techniques (PCR), and eight only through microbiological methods. Three studies analysed microbiological and epidemiological data. Only one article included a chemical identification analysis of antibiotics in samples in addition to molecular genetic analysis. All eligible research works addressed AMR in a rural site, but four studies also took samples in urban or peri-urban sites for comparison. In one study, we assumed the location was rural (poultry production sites), based on current poultry production practices, but it was not explicit in the article. As shown in Anthropogenic drivers of AMR included intensive and non-intensive  animal production systems ,49,50,51Poultry was the most common livestock ,49,50,52In contrast, Cicuta et al.  did not Armas-Freire et al. , BrisolaMore than half of the articles identified water as a contributing factor for AMR spread ,51,52,54Brisola et al. , CampionAs shown in This review identified key contributors to AMR in LA considering the OH concept. The following anthropogenic activities were identified as drivers for AMR dissemination in rural Latin American settings: animal husbandry, fish farming, agriculture, and other related practices such as animal waste recycling. The carriage and/or transfer of AMR determinants were the most frequent animal contributions, in addition to the inadequate or unregulated use of veterinary antimicrobials and the role of food of animal origin in the spread of AMR bacteria in the human food chain. Water was the most commonly identified environmental contributor for AMR spread but also soil, farm/bird coops, vectors (flies), and pond sediments were also important contributors mentioned.Nearly half of the eligible studies showed robust evidence confirming the human, animal, or environmental contributions to the generation or the spread of AMR. However, only one study  providedMost of the assessed articles studied the AMR problem from a single viewpoint or emphasised one of the OH components. Five studies ,47,48,49One of the most important insights from our study is the scarce research on humans or human waste as sources of AMR determinants in rural locations described in the Latin American literature. Studies focusing on AMR in rural health centres or rural hospitals were not found in the articles eligible for analysis. However, the impacts of rural hospital effluents on the environment and hence, on human and animal microbiomes cannot be ignored , mainly We found some information gaps in the selected literature. Mining is an important economic activity in many countries in LA  and it hWildlife is a neglected likely significant contributor to the spread of AMR in rural LA. Although it was not identified as a contributor in the eligible articles, the interaction between humans, wild animals, and farm and domestic animals occurs in rural settings, sharing AMR bacteria . Thus, sOnly one article used qualitative methods . QualitaOn the other hand, the most robust evidence for the role of animals, humans, and/or the environment in the spread of AMR originated from studies combining molecular methods, phenotypic resistance screening methods, sound study design and sampling, and an integrated OH perspective. A few studies relied upon a combined phenotypic resistance testing and an epidemiological approach, thus identifying risk factors and protective factors for AMR. However, due to the limitations of culturing in assessing AMR, they could not give insight into the specific AMR determinants associated with the AMR phenotypes . PhenotyIt is important to note that only one study in Brazil , mentionWe did not include grey literature, which we believe would have enriched our findings. There was no systematic way to search for country-level surveillance reports. Since most literature reviews only include publications in the English language, we purposedly looked for articles produced in LA written in Spanish and Portuguese languages, finding a considerable number during the screening process. However, none of them fulfilled the eligible criteria for this review. This limitation may be due to the kind of settings in which these studies have been conducted—urban as opposed to rural—and the AMR perspective applied, which may be one-sided, favouring any of the OH components but not comprising the three domains, as we specified in our inclusion criteria. Since AMR was recognised as a global threat to public health, virtually all countries adopted a national action plan to tackle the problem ; howeverOther reasons may explain the scarcity of truly integrated OH research works in LA—as in other low- and middle-income regions. Establishing OH research involves facing barriers such as lack of OH training and expertise, and difficulty in establishing collaboration among multiple and cross-sectoral actors—resulting in scarcity of multidisciplinary training programs—and limited government support and research funding ,71. FundThis scoping review on AMR in rural settings in LA identified the human, animal, and environmental contributions to AMR using an OH lens, and pinpointed the information gaps on AMR transmission routes and AMR drivers in the literature. Human activities contributed to the spread of AMR through animal husbandry (mainly poultry), fish farming, agriculture, and animal waste recycling (composting). Farm animals contributed by carrying and/or transferring resistant genes or resistant bacteria. Main environmental contributors are faecally-contaminated water, contaminated soil or pond sediments, and farm environments.Adopting an OH lens proved useful as a framework to determine whether the selected articles considered the impact of AMR on the three aspects of health, animal, human, and environment, and to what extent they did so. However, a small percentage of articles took into account the three OH components in the sampling or in the discussion. Thus, we recommend following the OH approach as a framework for the design of future studies—emphasising on the use of mixed methods and a combination of approaches—molecular-, epidemiological-, and culture-based—to tackle AMR more efficiently and to tailor strategies to prevent AMR emergence in the region, where these efforts are still scant and considerably needed.Future research efforts should give more attention to the role of mining, wildlife, and rural hospitals’ or health services’ effluents on the emergence and spread of AMR in rural Latin America, given that these aspects were not identified in the selected literature and were considered information gaps.


Tg) can be reduced by the incorporated PEG to enhance the ductility of the nanofibers. The SEM images and the MTT results demonstrated that composite fibers are suitable scaffolds for cell adhesion and proliferation. Dexamethasone (DEX), an osteogenic inducer, was loaded to PLA/MWCNT/PEG fibers. The surface element analysis performed by XPS showed that fluorine of DEX in pristine PLA fibers was much higher than those of the MWCNT-containing fibers, suggesting that the pristine PLA fibers mainly load DEX on their surfaces, whereas MWCNTs can adsorb DEX with evenly distribution in nanofibers. Drug release experiments demonstrated that the release profiles of DEX were manipulated by the ratio of PEG, and that the more PEG in the nanofibers, the faster DEX was released. When rat bone marrow stromal cells (rBMSCs) were seeded on these nanofibers, the Alizarin Red S staining and calcium quantification results demonstrated that loaded DEX were released to promote osteogenic differentiation of rBMSCs and facilitate mineralized tissue formation. These results indicated that the DEX-loaded PLA/MWCNT/PEG nanofibers not only enhanced mechanical strength, but also promoted osteogenesis of stem cells via the continuous release of DEX. The nanofibers should be a potential scaffold for bone tissue engineering application.Composite electrospun fibers were fabricated to develop drug loaded scaffolds to promote bone tissue regeneration. Multi-wall carbon nanotubes (MWCNTs) were incorporated to polylactic acid (PLA) to strengthen electrospun nanofibers. To modulate drug release behavior, different ratios of hydrophilic polyethylene glycol (PEG) were added to composite fibers. Glass transition temperature ( Due to the limited sources of autologous and allogeneic bone tissues as well as the possibilities of pathogen infection and rejection during transplantation, synthetic scaffolds such as bone substitutes have received considerable attention to replace and treat large-sized bone defects. Appropriate physical, chemical, and biological cues can be provided by these scaffolds to guide osteoprogenitor cells differentiation and promote bone regeneration . In ordePolylactic acid (PLA) is a Food and Drug Administration (FDA)-approved synthetic biocompatible material and can be electrospun as nanofibers . AlthougIn addition to mechanical support, scaffolds can also be applied as drug-carriers to regulate cellular physiology. Through the adjustment of scaffold composition, the delivery rate and duration can be delicately regulated to fulfill the therapeutic requirement during tissue regeneration. Dexamethasone (DEX) is a steroidal drug that is frequently used to reduce inflammatory response. Because DEX can regulate RUNX2, a critical transcription factor associated with osteogenesis, DEX accompanied with ascorbic acid and β-glycerophosphate is frequently used to guide bone marrow stromal cells to differentiate into osteoblasts and mineralization ,16. HowePoly (ethylene glycol) (PEG) is a hydrophilic polymer broadly used in pharmaceutical formation because it can facilitate drug-loading of hydrophobic carrier, such as PLA nanofibers . BecausePolylactic acid with molecular weight of 180~210 kDa is provided by Wei-Mon Industry Co. . Multi-wall carbon nanotubes (MWCNTs) are acid-oxidated to the level of COOH of 1.8 mol.% , and their diameters and lengths are between 10–20 nm and 10–30 μm, respectively. Polyethylene glycol (PEG) with molecular weight of 8 kDa, dexamethasone, sodium dodecyl sulfate (SDS), and thiazolyl blue tetrazolium bromide (MTT), pluronic F-127 are obtained from Sigma-Aldrich . Dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) are purchased from Thermo Fisher Scientific .Acid-oxidated MWCNTs were dispersed in DMF. To increasing dispersion homogeneity, SDS and pluronic F-127 in 10 wt % of MWCNTs were added to MWCNT solution , which wOn the other hand, 10 wt % of PLA and different amount of PEG were dissolved in DCM at room temperature. Then, the dispersive MWCNTs were dropwisely added to the PLA/PEG solution under ultrasonication. The details of the amounts of MWCNT and PLA solutions for the electrospun solution preparations are listed in To electrospin nanofibers, the polymer solution was fed to 1 mL polypropylene syringe with needle of 19G needle tip that was connected to a power supply . The working voltages of was 15 kV, and the feeding rate was 1 mL/h. To collect electrospun fibers, glass cover slips with diameters of 16 mm were warped on a grounded aluminum rotating mandrel that was rotated at 300 rpm. The working distances between the needle tip and the rotating mandrel collector was 12 cm. The electrospun nanofibers were dried in an oven for 1 day at 37 °C, and these prepared nanofibers would be applied as scaffolds for bone tissue engineering application.Morphology of electrospun nanofibers was examined by scanning electron microscopy , which were sputter-coated with gold before examination. The diameter distribution of nanofibers was calculated by 100 measurements based on SEM images. Regarding cell morphology, cells and nanofibers were fixed by glutaraldehyde and serially dehydrated by ethanol. Finally, specimens were lyophilized to completely remove moisture and gold sputtered before SEM analysis.The mechanical performance of the nanofibers was analyzed by a tensile test. Electrospun nanofibers were collected from the rotating mandrel, which were cut into rectangles with lengths of 3 cm and widths of 1 cm, where 3 cm was the side parallel to the circumference of the mandrel. These 3 cm fiber specimens were clamped on the tweezers of a universal testing machine  for 1 cm each end. Fibers were stretched in a constant speed of 3 mm/min. The displacement and corresponding pressure was continuously monitored until fracture .The hydrophobicity of the electrospun nanofibers was examined by a water contact angle analysis . Water drops were added to 4 points per sample, and their contact angles were presented as averages with standard deviations.−1 between 4000 and 600 cm−1. Surface chemical analysis of nanofibers was carried out by XPS . Peaks of C 1s and O 1s were resolvedly analyzed by iterative Gaussian/Lorentzian fitting .To determine the composition and functional groups of nanofibers, Fourier transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS) were performed. After electrospinning for 2 h, the prepared nanofibers were adhered to the sample holder of FT-IR spectroscopy . The measurement was in a resolution of 4 cmIn order to determine the response of nanofibers to temperature changes, DSC was applied for thermal analysis. We collected 6 mg of nanofibers and placed them in an aluminum pan. Then, the sample pans were evaluated by the DSC  to determine the difference in heat flow. The temperature was rise from 30 to 600 °C in a rate of 10 °C/min.The method of loading DEX to polymer solution for electrospinning was list in Rat bone marrow stromal cells (rBMSCs) were harvested from 8-week-old Sprague–Dawley rats . After b2. All experiments were performed triplicate. After 1-, 3-, or 5-day culture, 100 μL of MTT solution ) and 900 μL of medium were added to wells and kept for 3 h at 37 °C. The supernatant was removed and 1 mL of DMSO was added to dissolve formazan, which then was spectrometrically analyzed at the wavelength of 550 nm.To evaluate whether cells may adhere and proliferate on nanofibers, an MTT test was performed. Nanofibers were electrospun on round coverslips in diameters of 1.6 cm, and then were placed in 24-well plates. After sterilizing DEX-containing nanofibers by UV treatment for 1 h, rBMSCs were seeded to nanofibers in density of 17,000 cells/cm4 cells/well. Osteogenic medium was applied in this experiment, which was changed every other day. Because osteoblasts differentiated from rBMSCs can deposit calcium to form mineralized tissue, the calcium deposition was qualitatively examined by Alizarin Red S staining and quantified by the calcium ortho–cresolphthalein complexone method (Ca-o-CPC) assay reaction after being treated in osteogenic medium for 14 and 21 days . T. T34]. T−1, which is correlated to the stretching vibration of C-O in the terminal of PEG chains  of PLA, the ester group (O=C–O–C) of PLA, and the alcohol (C–O–H) as well as ether (C–O–C) groups of PEG, respectively  as a process called mineralization . The forx method d. When rActually, we also have seeded rBMSCs on nanofibers without DEX loading  and mineralization and calcium deposition cannot be found in these groups, suggesting that DEX delivery is essential to trigger rBMSCs osteo-differentiation (data not shown). The quantification results of mineralization indicated that differentiation of rBMSCs highly depends on the delivery profiles of DEX from nanofibers. Because DEX can control the activity of RUNX 2, which is a key transcription factor associated with osteogenesis ,16, the In this study, we successfully electrospun MWCNT/PEG/PLA composite nanofibers. These nanofibers were reinforced by MWCNTs, and the incorporation of PEG improved the ductility of nanofibers to avoid brittle break. In addition, these MWCNTs can adsorb DEX to facilitate the even distribution of DEX in the nanofibers. The duration and releasing rates of DEX release can be manipulated by the amount of PEG in nanofibers, i.e., as the concentration of PEG in nanofiber increased, the release of DEX was faster. Finally, the differentiation of rBMSCs on these fibers demonstrated that continuous release of DEX from the DL-0.5 C/0.1PEG and DL-0.5/1PEG groups effectively accelerated osteogenic differentiation and improved mineralization, suggesting the potential of these nanofibrous scaffolds for bone tissue engineering application.


There is increasing concern within the veterinary medical community (veterinarians and veterinary students) that disgruntled clients are unfairly leveraging various legal tools against veterinarians. Clinical veterinarians and veterinary students should be aware of the most common types of problems arising within the clinic and how they can lead to formal consumer complaints. The study describes and categorizes with greater detail the types of violations or “causes for discipline” that occur, as well as specific sanctions imposed on veterinarians formally disciplined for standard of care-related violations between 2017 and 2019, for California. In addition, the study calculated the frequency of disciplinary actions and their basic summary statistics regarding the temporal aspect of how lawsuits typically unfold. Using public documents from California, the study describes the analysis and trends for the purpose of providing contextual evidence to inform and guide potential veterinary educational interventions. Although specific to California, this study can serve as a template methodology for comparisons to other states. Increasingly, Americans view pets as important members of their social in groups rather than expendable property . Nonethevia the individual state's Veterinary Practice Acts (VPAs) and the state's Veterinary Medical Boards (VMBs). State VMBs oversee the practice of veterinary medicine to ensure both consumers and veterinarians are protected from, for example, substandard care or unsupported consumer complaints, respectively. VMBs and VPAs also ensure that the licensed veterinarian continues to uphold the Veterinarian's Oath. If a formal complaint is received, the state VMB uses its discretion whether to investigate and determine if disciplinary legal action is justified in accordance with state-specific laws. Disciplinary sanctions resulting from legal action also vary by state and can have serious adverse consequences for veterinarians, such as license revocation, probation, fines, and required continuing education  that disgruntled clients are unfairly leveraging various legal and social media tools against veterinarians . ClinicaBabcock et al.  attempteConsidering the differences in definitions to be a major limitation of the Babcock et al.  study, oTo obtain the data from formally disciplined veterinarians, a California Public Records Act (CPRA) request was performed on November 7, 2019, and approved by the California State VMB on November 15, 2019. The list we received was composed of veterinary license numbers for all veterinarians whose disciplinary action cases contained both a “Final Decision” and an “Accusation rooted in a sub-standard of care argument” between November 7, 2017 and November 7, 2019. Note that this population only includes disciplined veterinarians and not consumer complaints or investigations that failed to meet the VMB's criteria to pursue disciplinary actions. Consumers can initiate a complaint using the Department of Consumer Affairs, Veterinary Medical Board Consumer Complaint Form .via the License Lookup Portal tool built into the BreEZe database located within the California Department of Consumer Affairs (CA DCA) website. The disciplinary case documents collected consisted of two main types, Accusations or Decision and Order. These documents concern the bulk of the research focus and therefore constitute the primary data source used in this study.The list of veterinary license numbers was then used to identify and collect publicly available disciplinary case documents, All documents were downloaded, renamed according to case number and document type, and then uploaded to a secure, cloud-based working folder. Renamed files were structured systematically, so they could be precisely indexed within their associated folders. The case-retrieval document containing the list of veterinary license numbers was also stored within the digital database.Accusation documents, while data from the remaining variables was extracted from the Decision and Order documents , Ethics Training (ET), Community Service (CS), Clinical or Written Exam Status, Psychological Evaluation Status, and Drug or Alcohol Rehabilitation Status). Five variables  were derived secondarily. In other words, data for each variable was populated systematically as case content was reviewed. All case-related data was cleaned and cross-verified by another researcher to mitigate any potential data extraction inconsistencies.A comprehensive content review of five randomly selected cases was conducted to gain familiarity of legal document structure and to establish a preliminary data extraction procedure. Variable data were extracted directly from these documents and transferred to a novel spreadsheet, with individual veterinary disciplinary cases  along the vertical axis. Data from three variables  were extracted from the Notification Period, which indicates the number of days between the initial Trigger event and the day when the veterinarian was legally served notice of disciplinary action. Our definition of a Trigger event requires that the Accusation document included an incident date, incident location, and an objective description of an incident that ultimately led to a consumer complaint or unannounced premises inspection. The second time interval calculated is described as the Litigatory Phase. This was calculated by subtracting the Served date from the Order date. This interval captures the number of days between the veterinarian's notice of indictment and the exact date on which the VMB reached a disciplinary conclusion called the Decision and Order. The third time interval is described as the Total Resolution Time. Total Resolution Time was calculated by subtracting the initial Trigger event date from the Order date.Three distinct time intervals were calculated from dates extracted from documents. The first time period is described as the Sanctions imposed by the California VMB can be characterized into three categories, License-related, Time-Commitment, or Monetary. These three categories were then further described in terms of severity or frequency of occurrence. License-related sanctions include the following variables: Revocation Status, Suspension Status, Suspension Duration, Probation Status, and Probation Duration. Time-commitment sanctions involved the following variables: Continuing Education Status, Ethics Training Status, Community Service Status, Continuing Education Amount, Ethics Training Amount, Community Service Amount, and Continuing Education Topics. Monetary sanctions variables included the following: Restitution Status, Fine Status, Restitution Amount, Fine Amount, and Cost Recovery Amount.Accusation documents to identify and depict the geographic locations where disciplinary actions in our sample area arose. ESRI's ArcMap software (https://www.esri.com/en-us/home) was used to create a map document showing the spatial distribution of zip codes involved with the study.Zip code data was extracted from A second California Public Records Act request was performed on August 10, 2020, to obtain the annual number of consumer complaints against veterinarians, in addition to the annual number of active state veterinary licenses during the years 2017, 2018, and 2019. The California Department of Consumer Affairs responded by providing these data timely. Incidence rates were calculated for each individual year, as well as the 3-year average incidence rate to help understand the risk of veterinarians receiving a consumer complaint filed to the CA state VMB against their active license. This is distinguished from the remainder of the study where we are looking at only those complaints that result in disciplinary action.Causes for Discipline or Statutory Violations for each case were extracted from the Accusation and recorded in the profile for each case. The study focused on identifying and describing potential trends related to veterinary standard of care violations and their associated disciplinary sanctions. A tabulated data set was created for each disciplinary action case. RStudio Desktop (https://www.rstudio.com/products/rstudio/) was used to import, visualize, and describe the distribution of all quantitative variables in terms of basic descriptive statistics, such as central tendency and variance. For each disciplinary action case, the total number of Causes for Discipline, or Violations, was tabulated. From 59 veterinary disciplinary action cases reviewed, the study calculated the median, maximum, and minimum number of violations per disciplinary action case. A similar approach was taken with the imposed sanctions  along with the restitution and cost recovery. When the data was normally distributed a mean rather than median was reported. The resulting data was plotted using a box plot of the 25–75% interquartile range with whiskers to show minimum and maximum, and also inclusion of any outliers to the interquartile range on the displayed plots.The Throughout the 3-year retrospective study period from 2017 to 2019, the average incidence of formal complaints against veterinarians was ~7 complaints per 100 active veterinary licenses . SpecifiWhile all 59 of the veterinarians in the sample had their licenses revoked, this revocation was stayed for all but 16 veterinarians. In other words, 27% of formally disciplined veterinarians had their license outright revoked by the State of California. For the remaining 43 veterinarians (73%) who had their revocation stayed, this meant their revocation could be lifted if they fulfill various probationary contingencies.Of the 43 veterinarians who had their licenses stayed, contingent upon the conditions of probation, 23 veterinarians had their license suspended (made inactive) for a duration of time. For the 23 veterinarians who had their license suspended before they could start their probationary period, the median number of days their licenses were suspended was 20 days. The maximum number of suspension days was 120 days, while the minimum number of days was 3 days . FurtherOf the 43 licenses placed on probation, 14 veterinarians received community service mandates, 25 received ethics training mandates, and 41 received continuing education mandates. The median number of hours imposed for those who received community service, ethics training, and continuing education, was 20, 20, and 60 h, respectively. There were 3 outliers within the continuing education group, which skewed the data's calculated mean. There were also 3 outliers within the community service group, which skewed the mean higher –5.All veterinarians were required to pay the cost recovery for the investigation, excluding one veterinarian who paid $0. The median number of dollars veterinarians were required to pay for cost recovery was $9,600. The maximum amount was $64,456, while the minimum (excluding the veterinarian who did not have to pay the cost recovery) was $888. Of the 11 veterinarians required to pay restitution, the mean was $2,213, the maximum was $3,960, and the minimum was $1,000. Of the 33 veterinarians required to pay a fine, the mean was $2,424, the maximum was $5,000, and the minimum was $1,000 . These vEvaluation of the time-interval data found that the mean number of years for the notification period, the litigatory phase, and the total resolution was ~3, 1, and 4 years, respectively.From the sample of 59 veterinary disciplinary action cases, the study calculated the median, maximum, and minimum number of violations per disciplinary action case . The top three most common types of violations were professional negligence (76%), records-related (66%), and incompetence (46%). Although many of the violations and decision orders were detailed and specific to instances of violations, some general examples provide guidance on behaviors or actions that should be avoided.For example, under a records-related violation, the veterinarian did not keep any medical records whatsoever for an unknown number of patients. When an unsatisfied owner requested medical records, the veterinarian failed to provide any documents to the client, and then the client proceeded to file a complaint with the VMB. When the veterinarian was subpoenaed by the VMB, the veterinarian fraudulently forged medical records to submit for review.As an example of incompetence violation, one veterinarian spayed a service dog in estrus and left the dog alone in a cage to recover overnight, and the dog was found deceased the next morning. A necropsy revealed the animal died from uncontrolled intra-abdominal hemorrhage, secondary to inadequate ligation of an ovarian pedicle.Finally, in one case involving professional negligence, a veterinarian anesthetized a cat prior to performing dental extractions and then the veterinarian left the surgery area. After waiting 20 min to start the procedure, a technician found the veterinarian unresponsive from a drug overdose in the apartment located above the clinic.Some notable patterns were identified from our statistical analyses. All VMB disciplinary actions involved feline, canine or a combination of these species. Two disciplinary actions also included pet porcine as patients. Surprisingly, no disciplinary cases involved client complaints with horses or livestock. The zip code analysis suggests that urbanized areas with greater population density experienced a greater number of disciplinary actions . HoweverN = 59) consisted of all Disciplinary Actions rooted in a standard of care argument that conclusively reached a Final Decision and Order between the years 2017 and 2019 in the State of California, which represents <5 cases per 1,000 active licenses. The data represented veterinarians who had at least one complaint filed to the VMB which ultimately resulted in a VMB disciplinary action with at least one or more sanctions. One important limitation to understand regarding the data and analysis in this study is that our statistical results do not represent all complaints filed against the veterinarians during this period nor all the details from the subsequent investigations. Rather, the data is from complaints and investigations within the VMB public documents which were used to support the disciplinary measures imposed by the VMB. Therefore, consumer complaints or investigations that failed to meet the VMB's criteria to pursue disciplinary actions were not included in the in-depth study. Informal complaints and complaints without sufficient justification for disciplinary action are more common, but difficult to study systematically.Overall, complaints to the CA VMB are very rare with only 7 in 100 active licenses having complaints filed. Our sample  and restitution and cost recovery related to the disciplinary action. In the box plotted data, the few outliers beyond the maximum range represent veterinarians with more egregious and intentional acts such as fraudulently altering the medical record, obtaining blood from euthanized or dying patients without client consent, not cross matching or testing the blood for blood borne diseases, or falsifying health certificates. Additionally, prior citations from the VMB were noted in the findings and were considered aggravating elements.However, this study revealed that the topic of veterinary disciplinary action related to standard of care violations is extremely nuanced and would be extremely challenging to assess/categorize at the national level until appropriate methods for state-level analyses are established. The terms, documentation components, and code sections used by state VMBs can vary between states. One question our study sought to answer was whether this approach can be scaled-up to compare state differences, and we found creating a uniform objective data entry spreadsheet was challenging to create when comparing records from California to Ohio for those reasons listed as well as available access to the public records during COVID-19 pandemic imposed closures. Moreover, to directly compare California and Ohio, some subjective interpretation of matching code sections was required; therefore, the analysis was not pursued. Until a national legal framework is created, this seems unlikely. Because the AVMA's Model Veterinary Medicine Practice Act merely provides unifying recommendations to state VMBs, the legal framework for veterinary law will continue to be extremely variable from state to state, preventing state-to-state comparisons from being possible or appropriate.record keeping continuing education requirements. One hypothesis that could explain why record keeping violations and related sanctions were common findings, is that medical records are subpoenaed legal documents which can serve as hard, indisputable, time-stamped evidence of professional negligence in an otherwise “factually” weak disciplinary argument. In other words, there might have been more serious violations involved in a disciplinary case, but there was no or insufficient evidence to support prosecution of those violations.Approximately 66% of all cases cited professional negligence related to record keeping. It is not surprising that about 44% of all cases requiring probationary conditions also included mandates to fulfill Notification Period highlights that veterinarians might not know for several years that their actions/behaviors might become subject to disciplinary action(s). The Litigatory Phase duration suggests that once a veterinarian is legally served, a disciplinary order (when justified) is mandated more quickly . However, it should be noted that the true amount of time involved in a disciplinary action case is much longer, since probation can last for many years beyond sentencing or if a veterinarian seeks to appeal the VMB finding then it will also result in a longer case duration. The study did not track the fulfillment status or sanction completion of veterinarians on disciplinary probation, and this additional time was not included in this study's time-interval analysis. The Total Resolution Time suggests that the overall litigative process is lengthy, often lasting more than 4 years.Other notable patterns described concerns the time-interval data and the time duration required for these cases to typically unfold and come to a final decision regarding the sanction. The implications of the longer-than-expected Perhaps the most meaningful conclusion from this study is that more stringent and consistent record keeping behaviors could confer significantly increased legal protection from disciplinary actions. While there is a potential for veterinarians to spend too much effort on record keeping such that they diminish their ability to provide care, our analysis suggests that legal action still relates more toward lack of records than over-recording. More research regarding record keeping as a potential target for educational intervention is needed. As the field of veterinary medicine continues to evolve we expect record keeping and documentation to become more important, especially in the areas of antimicrobial use, pandemic-related waivers for telemedicine,The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The project was conceived by AM and RG. AM prepared the funding proposal. JL and RG designed the data entry and subsequent analysis. JL and CS collected and collated the results. The manuscript was prepared by JL and reviewed by all authors with significant editorial assistance from AM.Support for this study was provided by the Stanton Foundation, a private foundation with canine health and welfare as one of its primary missions.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.


To use the example of COVID-19 vaccine prioritization for incarcerated workers to call attention to the need to prioritize incarcerated workers’ health.From November to December 2020, we searched publicly available information (e.g. Department Of Corrections websites and press releases) for 53 US prison systems, including all states, Immigration and Customs Enforcement, the Federal Bureau of Prisons, and Puerto Rico. Coders reviewed if states had prison labor policies, if states had COVID-19 specific prison labor policies, the location of work, industries both pre- and during the COVID-19 pandemic, the scope of work, and hourly wage. Findings were compared to the Centers for Disease Control and Prevention’s occupational vaccine prioritization recommendations.Every facility has incarcerated individuals working in some capacity with some resuming prison labor operations to pre-pandemic levels. All but one prison system has off-site work locations for their incarcerated population and many incarcerated workers have resumed their off-site work release assignments. Additionally, every state has incarcerated workers whose job assignments are considered frontline essential workers (e.g. firefighters). In at least five states, incarcerated workers are participating in frontline health roles that put them at higher risk of acquiring COVID-19. Yet, no state followed the Centers for Disease Control and Prevention recommended vaccination plan for its incarcerated population given their incarcerated workers’ essential worker status.The Centers for Disease Control and Prevention recommended that incarcerated people be prioritized for vaccination primarily due to the risk present in congregate style prison and jail facilities. Furthermore, our review found that many incarcerated people perform labor that should be considered “essential”, which provides another reason why they should have been among the first in line for COVID-19 vaccine allocation. These findings also highlight the need for incarcerated workers’ health to be prioritized beyond COVID-19.The online version contains supplementary material available at 10.1186/s12889-022-12886-7. The Centers for Disease Control and Prevention’s (CDC’s) Advisory Committee on Immunization Practices (ACIP) listed people who are incarcerated at increased risk of COVID-19 acquisition and transmission and has thus identified them as a critical population deserving of COVID-19 vaccine prioritization .One of the four risk categories to determine vaccine prioritization is the risk of negative societal impact . This crIncarcerated workers are an overlooked population in workplace protections even though half of those incarcerated in federal and state prisons work full-time . For exaThe 13th Amendment outlawed slavery except as a punishment for crime, which enables incarcerated individuals to be forced to work as part of their sentence without proper workplace protections , 8. GiveUsing prison systems’ department websites, press releases, social media pages, and news articles, we identified whether each of the 53 prison systems has a prison labor policy, a COVID-19 specific prison labor policy, the location of work, industries both pre- and during the COVID-19 pandemic, the scope of work, and hourly wage. This data was collected between November and December 2020.Two coders independently searched for information and a third coder reconciled differences. The finalized industries were compared to the CDC’s recommendation for occupational vaccine prioritization to identify which states have listed incarcerated individuals as essential workers and the relevant corresponding vaccination phase 1A, 1B, 1C) . Phase 1A, 1B, 1CThe full data from this research is available in the attached supplemental material  have confirmed that incarcerated workers may be exposed to COVID-19 through work in mortuary services and cleaning hospital laundry.Every state has incarcerated workers whose job assignments are considered frontline essential workers under Phase 1B. The most common industries for incarcerated workers were manufacturing, agriculture, firefighting, and meat processing. Twelve states have incarcerated workers who participate in other essential services such as food services and construction.Every state has a prison labor policy, however, 18 states  did not have a COVID-19 specific prison labor policy. Many states pivoted to manufacturing personal protective equipment and increased sanitation jobs due to the COVID-19 pandemic. Only three systems  made specific mention of social distancing in their COVID-19 specific prison labor policies. Three systems  moved incarcerated workers into the same housing unit based on work assignments. However, it is unclear to what extent these policies are enforced.Incarcerated workers have continued working both on-site and off-site at the federal and state levels. During our search in November to December 2020, twelve systems had suspended all off-site work details in their most recent policy update, while the other states had continued or resumed.There is significant variation in hours across systems and wages. Most facilities have a maximum of forty hours a week in line with full-time work in the general public. However, some allow up to 60 h a week or 12 h a day. Many states required deductions from wages for court debts, taxes, mandatory savings, or charge for custody and care . Only those working in mortuary services have been given pay increases for COVID-19 facing work. In New York, workers transitioned from an average hourly wage of 65¢ to $6 for digging mass graves. In Texas, workers who were previously unpaid received $2 per hour to work in the mobile morgues.Prior literature has advocated for incarcerated individuals being prioritized for COVID-19 vaccinations given the high-risk environments of congregate, over-crowded settings . There hWe argue that in addition to prioritizing COVID-19 vaccination for all incarcerated persons, incarcerated workers must be prioritized in alignment with their job. While recent efforts, such as in New York, have sped up the vaccination of incarcerated individuals, the prioritization of incarcerated workers specifically has not occurred .The main limitation of this study was relying on publicly available information as we are unable to verify the implementation of COVID-19 labor policies in carceral settings and the collected data is not specific enough for differentiating between occupations and industries. We found examples of changes in prison labor protocols without an update on COVID-19 prison labor policies. Further research should involve interviewing incarcerated workers on their experiences working before and during the pandemic. Additionally, COVID-19 vaccine prioritization and roll-out are ever-changing. It is possible that our information is out-of-date at the time of publication. Nonetheless, it is important to highlight that as vaccinations were approved and prioritization was assigned at the end of 2020, incarcerated workers were absent from state plans.This research further illustrates the already high need for incarcerated individuals to be prioritized for the COVID-19 vaccine as many incarcerated individuals work at jobs that are high risk for transmission and exposure to COVID-19. This research also highlights that incarcerated workers’ voices are not typically included in workplace safety or occupational health surveillance tools like OSHA complaints. Future research is needed for the assessment of prison labor policies and health outcomes during and beyond the COVID-19 pandemic at the prison system level.Furthermore, to better prepare for future health crises and potential vaccination campaigns, incarcerated workers must be fully recognized as essential workers and be prioritized as such.Additional file 1.