One fundamental strategy to address the public health threat of antimicrobial resistance (AMR) is improved awareness among the public, prescribers, and policy makers with the aim of engaging these groups to act. World Antimicrobial Awareness Week is an opportunity for concerted and consistent communication regarding practical strategies to prevent and mitigate AMR. We highlight 10 ways for antimicrobial stewards to make the most of World Antimicrobial Awareness Week. Currently, global statistics estimate that 1.27 million deaths are directly related to AMR annually. Moreover, AMR extends beyond increased health risks to agriculture, food security, and socioeconomic development, making it one of the world’s most urgent public health issues. To bring about needed change, the World Health Organization (WHO) has developed a Global Action Plan on Antimicrobial Resistance. The first of 5 objectives of this plan is “to improve awareness and understanding of antimicrobial resistance through effective communication, education and training.” Awareness is the initial step toward ensuring the continued ability to treat and prevent infectious diseases worldwide. In this article, we convey key considerations when developing antimicrobial awareness campaigns, with a particular focus on World Antimicrobial Awareness Week, while highlighting relevant work published in Antimicrobial Stewardship & Healthcare Epidemiology (ASHE).Antimicrobial resistance (AMR) endangers health by its effect on morbidity, mortality, hospital length of stay, and healthcare costs.1. WAAW promotes best practices among the public, clinicians, and policy makers to reduce AMR. The WHO encourages the promotion of optimal antimicrobial use and preventive measures targeting AMR, collaborating across sectors and borders, and recognizing the interconnection between humans, animals, plants and the environment, a concept known as One Health.World Antimicrobial Awareness Week (WAAW) is a global event held annually from November 18–24.2. Examples may include direct communication of local prescribing rates and antibiograms (if available) to practitioners and public education to reduce antibiotic misuse for viral infections. Countries’ socioeconomics may also influence the messaging, such as minimizing access to antibiotics without a prescription in low- and middle-income countries and reducing sharing of antibiotics or the use of shorter courses in high-income countries.Most campaigns have generic messaging targeting the awareness of healthcare workers and the public. But adapting messaging to unique local issues may help close knowledge gaps or address different cultural beliefs related to antibiotics.3. Both clinicians and patients view AMR as a geographically and temporally distant phenomenon with a less personal impact on them. Rather than the more nebulous societal consequences of AMR, antimicrobial awareness campaigns should shift focus to the personal impact of AMR, including the very tangible and current risk of side effects, personal risk of AMR affecting future treatment options, and the perturbations to the microbiome.While campaigns focusing on the approaching ‘antibiotic apocalypse’ work to capture public attention, qualitative interview data show that these messages are often too sensationalized, which may backfire on their credibility.4. Campaigns like ‘Go Blue for AMR’ include wearing light blue and illuminating local buildings and landmarks in light blue, which can further the visibility of what some have called the ‘silent pandemic.’What does AMR look like? The concept of AMR is abstract to many. The exceedingly large projections of the impact of AMR do not tell the personal story of the devastation caused by drug-resistant infections on individual lives. Sharing patient stories can help ‘put a face’ to AMR, making the problem more relatable and inspiring efforts for change.5. Of those antibiotics, 20%–50% are used inappropriately. Primary care providers cite patient pressure for prescribing antibiotics for viral illnesses. Engaging physicians, veterinarians, and other healthcare professionals and policy makers in AMR awareness is essential to promoting One Health coalitions. This need is highlighted by the WAAW 2022 theme, ‘Preventing antimicrobial resistance together.’ Professional education and training for students and practitioners in human and animal health should also be established, as should the inclusion of antimicrobial use and resistance in school curricula and media to promote a better understanding of AMR.Antimicrobial stewardship was initiated in the hospital setting; however, 80% of antibiotics are used in the community. found that while ambulatory cancer center staff largely knew the term “antimicrobial stewardship,” there were opportunities to improve knowledge about appropriate antibiotic use, particularly among nurses and other clinic staff. Catanzaro et al highlighted the importance of involving nurses in antimicrobial stewardship using educational modules. Similarly, Manning et al demonstrated how patient simulations can improve nursing-student awareness of their role in antimicrobial stewardship. Hughes et al identified several opportunities for improvement in dental antimicrobial stewardship, including improved awareness of and access to evidence-based guidelines, as well as harnessing social comparison to improve prescribing behavior.Several recent ASHE publications have stressed the importance of broadening the horizon of antimicrobial stewardship awareness beyond prescribers and patients as well as outside the hospital walls. Gullen et al6. In a retrospective cohort study, 205 patients (80.7%) received empiric antibiotic therapy indicated for presumed urinary tract infection despite meeting criteria for asymptomatic bacteriuria. However, given the known harms of antibiotic therapy, there is an opportunity to reframe messaging regarding “erring on the side of caution” to now mean thoroughly evaluating and monitoring the patient while considering other noninfectious causes before prescribing an antibiotic.Antibiotics are often prescribed “just in case” and to “be on the safe side.” In a cross-sectional study using interviews of 90 dentists, 91.2% stated that they prescribed on a ‘just in case’ basis.7. but at the same time, it provides an instant connection between infectious diseases experts and the public. Social media platforms present not only an opportunity, but a responsibility to share accurate, clear, consistent, and engaging messaging on AMR. Endless opportunities exist to engage healthcare professionals and the public using creative approaches such as infectious diseases memes, clue-based knowledge assessment quizzes, and personality quizzes. Antibiotic awareness messaging can be embedded in each engagement strategy, tailored to the captive audience.Social media has facilitated the rapid spread of health-related misinformation,8.Antibiotics don’t treat viral infections, whether it be COVID-19 or the common cold. In the United States, ∼44% of outpatient encounters include acute upper-respiratory infections. Despite the majority of illnesses being caused by viruses, many patients still receive antibiotics, suggesting a need for improved awareness of the risks and benefits of antibiotics in this setting.Infectious diseases can be devastating. Unlike COVID-19, AMR is not caused by a single organism and presents a more insidious threat. We need to act quickly to prevent these devastating consequences while we still can. The increased awareness regarding the importance of infection prevention and control during the pandemic should also apply to AMR and may help garner research funding and public-sector support.The lessons we learned during the COVID-19 pandemic can also apply to antimicrobial stewardship.9. In addition, behavioral science needs to be incorporated into campaigns and formally evaluate and disseminate findings to help inform future antimicrobial awareness activities. Of 60 campaigns surveyed by the WHO, only 25 were evaluated, and for most of these campaigns, the results were not published. Wellcome’s Reframing Resistance report provides a list of practical evidence-based suggestions for awareness campaigns, including shifting fear-based messaging to that focusing on personal impact and opportunities for immediate action. Although there is evidence that fear-based messaging can be effective, it should be combined with empowerment to encourage action rather than apathy.Antimicrobial awareness messages should be based on scientific evidence. A previously common message to “finish the antibiotic course, even if you are feeling better” was replaced with more nuanced messaging recognizing the growing evidence base for shorter and more tailored courses of therapy.10. Thus, World Antimicrobial Awareness Week should be considered the first step in a long and iterative process, combined with policy, economic, and social strategies to improve antibiotic use.Antimicrobial awareness should not end after WAAW. Awareness of AMR is necessary but insufficient to change behavior. The impact of awareness campaigns on antibiotic use is mixed, but any impact will not be sustained without ongoing messaging.In conclusion, AMR is a global and universal issue, neither respecting borders nor confined to certain populations. AMR already has a significant impact on health, and if no action is taken, it will become increasingly burdensome. AMR is a solvable issue and awareness of antimicrobial overuse and its consequences is the first step to safeguarding antimicrobials. The 10 concepts discussed in this article can be applied to help maximize antimicrobial awareness efforts as a vital step in ensuring that we can continue to treat and prevent infectious diseases in the years to come.


Myocardial infarction is still a life-threatening disease, even though its prognosis has been improved through the development of percutaneous coronary intervention and pharmacotherapy. In addition, heart failure due to remodeling after myocardial infarction requires lifelong management. The aim of this study was to develop a novel treatment suppressing the myocardial damage done by myocardial infarction. We focused on inhibition of soluble epoxide hydrolase to prolong the activation of epoxyeicosatrienoic acids, which have vasodilatory and anti-inflammatory properties. We successfully made a new vaccine to inactivate soluble epoxide hydrolase, and we have evaluated the effect of the vaccine in a rat myocardial infarction model. In the vaccinated group, the ischemic area was significantly reduced, and cardiac function was significantly preserved. Vaccine treatment clearly increased microvessels in the border area and suppressed fibrosis secondary to myocardial infarction. This soluble epoxide hydrolase vaccine is a novel treatment for improving cardiac function following myocardial infarction. In particular, percutaneous coronary intervention dramatically improved mortality from myocardial infarction2. However, the number of patients with heart failure (HF) associated with myocardial infarction (MI) has been increasing as the number of MI survivors increases. Therefore, it is important to minimize the extent of myocardial damage by MI to suppress myocardial remodeling and prevent the development of HF.Cardiovascular disease (CVD) is still a major cause of mortality worldwide3. These metabolites are known to potently promote anti-inflammation, vasodilation, fibrinolysis, anti-apoptosis, and angiogenesis4. We have been focused on EETs as promising candidates for cardioprotection since Koichi Node et al. reported their anti-inflammatory properties in addition to their vasodilatory action5. Gross et al.6 reported that EETs reduced ischemic size and area at risk in a myocardial ischemia model. Because soluble epoxide hydrolase (sEH) very quickly metabolizes EETs into dihydroxyepoxyeicosatrienoic acids (DHETs) that are not cardioprotective, several approaches have been made to enhance the activity of EETs by inhibiting sEH. Ex vivo studies in mice have demonstrated significant cardioprotective effects of not only EETs but also sEH inhibition against ischemia–reperfusion injury involving the PI3K pathway8.Epoxyeicosatrienoic acids (EETs) are cytochrome P450 2J2 epoxygenase metabolites of arachidonic acid7. Furthermore, sEH inhibitors blocked NF-κB activation and reduced cardiac hypertrophy in the transverse aortic constriction (TAC) model of pressure-overload hypertrophy in mice9.Inhibition of sEH also exerted beneficial effects on cardiac function and post-MI ventricular remodeling, with direct positive effects on fibrosis and hypertrophy10.However, there are as yet no clinically approved sEH inhibitors for use in humans. Some candidates had poor metabolic stability, a relatively high melting point, and limited solubility in water, properties which made them difficult to use pharmacologically11. However, its cardiac protective effect was comparatively modest, being less effective in reducing cardiac hypertrophy in the TAC model12.Recently, the novel sEH inhibitor GSK22562940 proved to have a physiological effect, producing increased endothelial vasodilatory functionTherefore, we decided to develop a vaccination system to inhibit the ability of sEH to reduce EETs activity. This study confirms that the sEH vaccine induced the production of antibodies in rats that inhibited sEH and increased the ratio of EETs to DHETs. The extent of myocardial infarction was correspondingly reduced in the vaccinated group, demonstrating the vaccine’s myocardial protective effect.o 7-wk-old male C57BL/6NCrslc mice four times at two-week intervals  as a carrier protein to induce the specific anti-sEH antibody. Knowing the sEH epitope information allowed three candidate peptides to be selected as antigens . The sEH vaccine (6.25 μg/ rat) (n = 4) was administered to each rat at 5, 7, 9, and 11 weeks of age  or sEH vaccine (n = 30). Rats that died within 24 h were surgery-related deaths, and rats that did not show an EF reduction of 15% or more immediately after surgery were excluded from the study. Three rats died after 24 h, two in the saline group and one in the vaccine group. We analyzed a total of 51 rats that remained alive on day 7 after MI induction . Transthoracic cardiac echocardiography was used to evaluate the effect of the sEH vaccine at baseline, 1 day and 7 days after MI induction  and fractional shortening (FS) were also not different at baseline and day1 after MI induction  in the vaccinated group was smaller than in the saline group. The left ventricular anterior wall (LVAW) in the saline group was thinner than that in the vaccinated group, indicating that the sEH vaccine significantly preserved LVAW after MI (P < 0.05). Both FS and EF were increased in the vaccine group compared to the saline group (P < 0.05).Taken together, the sEH vaccine suppressed the dilation of the left ventricle due to myocardial infarction and improved systolic function.Azan staining in the short axis of cardiac muscle showed that infarct-induced fibrosis was significantly lower in the vaccinated group than in the saline group  in the border area were randomly chosen and the labeling densities were measured.Intramyocardial arteries and capillaries in the border zone on 7 days after MI were assessed. For labeling, we used lectin specialized to bind specific oligosaccharide side chains of vascular endothelial cells Fig. a, anti-CThe number of myocardial vessels including capillaries and arteries in the sEH vaccine group was significantly greater than that in the saline group, suggesting that sEH inhibition promoted angiogenesis in the border area between the ischemic and non-ischemic areas.14. In confirmation, the protein expression level of VEGF in the vaccinated group was increased compared to that in the saline group -ethoxy]-pentyl}-urea (AEPU) and trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), successfully reduced infarct size and inhibited the progression of cardiac remodeling in an ischemia–reperfusion model by suppressing inflammation31. Another sEH inhibitor, 12-(3-adamantane-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), also reduced ischemic injury in an ischemic-reperfusion model in mice32 even though it was administered intraperitoneally. Our results showing that the sEH vaccine attenuated myocardial infarction are consistent with those preceding reports.Several reports have shown beneficial effects of increasing EETs in mouse models of LAD ligation. The perfused hearts from sEH-null mice showed greater recovery from LAD ligation for 20 min than those from wild-type mice through PI3K-GSK-3β signaling and KThe difference between these publications and our experiments is the accessibility of the sEH by the sEH inhibitory agents. Because sEH is known as an intracellular protein, it is thought to be difficult to for the neutralizing antibody access sEH protein directly though membrane of the lipid bilayer. We postulated that cell membrane could loose and sEH was leaked from the cardiac muscle when the cells went necrosis as well as creatine phosphokinase, troponin, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase during process of MI. In fact, we found that sEH were diffusely stained in the cardiomyocytes  in many vascular beds, including the coronary artery and renal circulation, and lowers blood pressureFor clinical application in humans, after first confirming that antibodies capable of inhibiting sEH can be raised in human blood, patients with overlapping risks of atherosclerosis, such as those who are prone to myocardial infarction due to diabetes, hypertension, and/or obesity, may be eligible for the sEH vaccine. In addition, since those who have had a previous myocardial infarction are prone to recurrence, sEH vaccination at the time of the first event may be cardioprotective. Vaccine effects are maintained far longer compared with oral dosing of conventional drugs, and the vaccine could be purified at a lower cost than long-term use of conventional drugs.In conclusion, we have demonstrated that the sEH vaccine has a protective role in acute myocardial infarction, showing reduced ischemic area and fibrosis. The sEH vaccination upregulates VEGF and increases vascularity in cardiac muscle after MI in rats. We propose that the sEH vaccine is a novel candidate for a preventive treatment to reduce myocardial damage after MI.27. We used these vaccines with complete Freund’s adjuvant for the first vaccination, and we additionally injected the vaccines with incomplete Freund’s adjuvant three times at two-week intervals . The sEH peptide vaccines consisted of 0.13 mg/mL sEH peptide and 0.87 mg/mL KLH in sterile PBS.Based on high antigenicity analysis, we predicted qualified peptide structures from epitope information. We selected one of three different antigenic peptides from the amino acid sequence of mouse sEH . After Cys was added to the N-terminus of each peptide, N- succinimide was used to conjugate each peptide to KLH 29. The candidate antigenic peptide-BSA conjugate  was coated at a 10 μg/ml concentration and diluted in 50 mM carbonate buffer overnight at 4 °C on 96-well ELISA plates . After blocking with PBS containing 5% skim milk, the sera were serially diluted in blocking buffer, added to each well, and incubated overnight at 4 °C. After washing each well with 0.05% PBS Tween-20 (PBS-T), the cells were incubated with horseradish peroxidase-conjugated antibodies specific for mouse IgG  for mouse serum, or with rat IgG  for rat serum, for 3 h at room temperature. After washing the wells with PBS-T, color was developed with the peroxidase chromogenic substrate 3,3’,5,5’-tetramethylbenzidine , and the reactions were terminated with 0.5 N sulfuric acid. The absorbance was measured at 450 nm using an Immunomini NJ-2300 . The half-maximal antibody titer was determined according to the highest value in the dilution range of each sample.The serum sEH antibody titer was quantified by ELISA as previously reported2O was added to an equal volume of hemocytes to hemolyze and 30 uL of plasma was also added. 14,15-EET-d11  11,12-EET-d11 (Item No.10006413) and 8,9-EET-d11 (Item No. 10009998) at 2.2 ng/μL , and vortexed. After 15 min incubation at 37 °C, 3 mM zinc sulfate was added to stop the reaction and samples were preserved at − 20 °C. Stocked samples were extracted three times with the same amount of ethyl acetate. The organic phase was collected and evaporated by miVac Duo .Rat plasma including antibodies that were preserved at the day7 from LAD ligation was used. The hemocytes including rat sEH protein were collected by fresh blood from one alive rat. 30 μL of dHWe used LCMS-8030 . A Sim-pack XR-ODSIII 2.2 μm C18 150 mm × 2.0 mm HPLC column  was used for HPLC separation. The column was kept at 40 °C. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. LC gradient was as follows: 45%B → 75%B (15 min) → 75%B (15 min) → 45%B (15 min) → 45%B (10 min). The flow rate was 0.2 mL/min. A typical injection volume was 18 μL using partial loop injection mode.We used LCMS-8030 with an electrospray ionization interface (ESI), operating in negative mode. The instrument was optimized by infusing a 0.1 μg/mL solution of 14,15-EET-d11 and 14,15-DHET-d11  in acetonitrile at 10 μL/min with a flow of 0.2 mL/min 25/75–0.1% formic acid in water/acetonitrile from the LC compartment into the mass spectrometer. The multiple-reaction-monitoring (MRM) transitions of m/z 330 → 219, and m/z 348 → 207 were chosen for 14,15-EET-d11 and 14,15-DHET-d11, respectively. We selected the MRM transitions from signal to noise ratio and selectivity. The MRM transitions for 14,15-DHET-d11 were acquired at 1.7 min. MRM transitions for 14,15-EET-d11 were acquired at 2.5 min. The DL temperature was 300 °C. Data were processed using Labsolution software . Calibration plots of 14,15-DHET-d11 and 14,15-EET-d11s were constructed. The ratio of 14,15-EET-d11 to 14,15-DHET-d11 in each sample was calculated.All experiments followed the ARRIVE guidelines and were performed in accordance with the “Position of the American Heart Association on Research Animal Use” and approved by the Institutional Animal Care and Use Committee at Saga University. Wistar rats were purchased from Japan SLC. Rats were randomly assigned to two groups: a saline group (n = 40) and a vaccinated group (n = 30) for MI evaluation, and a saline group (n = 4) and a vaccinated group (n = 6) for vaccine safety evaluation. Rats were vaccinated a total of 4 times, on weeks 0, 2, 4, and 6, while 100 μL of saline was injected into the non-immune saline group rats.On week 8, rats were anesthetized with Nembutal (100 ml/kg) and intubated for mechanical ventilation. We performed a left thoracotomy to expose the heart and ligated the left anterior descending artery with 6–0 Prolene suture to create an MI; echocardiography was carried out immediately after surgery to verify MI creation. We selected cases based on a 15% or greater reduction in EF compared to preoperative EF.One week later, we harvested the heart and other organs to examine the effects of the vaccine treatment.A tail-cuff system  was used to measure blood pressure and heart at 11 weeks of age in rats without MI induction to evaluate the safety of the sEH vaccine. The rats were allowed to adapt to the apparatus for more than one week before the study.34, we measured the thickness of the anterior wall, diastolic dimension of the left ventricle (LVDd), and LVDs. FS (%) was calculated as (LVDd-LVDs)/LVDd × 100%. The left ventricular EF (%) was calculated as [(LVDd)3 − (LVDs)3/(LVDd)3 × 100%]. Similar and different parts of the heart were measured twice, and averaged data were obtained.Transthoracic echocardiography was performed in the rats before and after surgery on day 0 and day 7 using an echocardiography machine with a 15-MHz transducer . M-mode was used for left ventricular echocardiographic recording. Using 2-dimensional short-axis imaging with the M-mode as described previously42 was calculated with ImageJ43 and taken as percent fibrosis. The investigators were blinded to treatment.Hearts were harvested from the rats after the echocardiogram examination at one week after MI (day 7). The hearts were fixed in formalin, and sections 2 mm below the LAD ligation level were Azan-stained to make fibrotic areas appear blue Fig. . The ratGriffonia simplicifolia Lectin I- Isolectin B4 (Lectin)  was used to visualize endothelial cells. Hoechst 33342 dye  was used to stain nuclei. We analyzed these data with HALO AI .Antigen retrieval was performed in Dako pH 9 EDTA buffer  with a microwave oven and a pressure cooker. Blood vessels were observed by staining for vascular endothelial cells and smooth muscle, with CD31  and α-smooth muscle actin (α-SMA)  , respectively. 28.In addition, we evaluated the safety of the sEH vaccine in rats without MI induction with histopathology. The lung, liver, and kidney were harvested from both control and vaccinated rats at 13 weeks of age. We stained them with hematoxylin and eosin after formalin fixation to determine whether there were adverse pathological changes in the vaccinated groupHeart sections were homogenized in RIPA buffer with protease inhibitors. Samples of heart lysates were resolved on SDS-PAGE according to a standard protocol. After transferring protein to membranes, the membranes were probed with primary antibodies followed by secondary antibodies conjugated to horseradish peroxidase, and immunoreactive bands were developed with ECL Plus Western Blotting Detection Reagents . Enhanced chemiluminescence was detected with FUSION FX , and band density was quantified with Image J software. We used the following primary antibodies: β-actin I-19  and VEGF (A-20)  from Santa Cruz Biotechnology , and eNOS  from Cell Signaling Technology .t-test. All statistical analyses were performed using Excel and JMP software programs .All data are expressed as mean ± SEM. Statistical analyses were conducted by one-way analysis of variance followed by Tukey's test or a two-tailed Student's Supplementary Information.


Endemic Kaposi’s sarcoma (KS) remains a major public health concern in Eastern and Central Africa, and diagnostic and therapeutic management represents a great challenge in a setting of financial limitations in resource-poor environment. Factors arising from specific ethnic behavior and geographical variation are responsible for the development of KS in the African population infected with HHV-8 mostly through salivary transmission. Our review provides an overview of the clinical characteristics and discusses the therapeutic challenges of this subtype. As endemic KS may follow an aggressive course, it often requires the use of systemic therapy. Immune checkpoint blockade could represent a promising alternative for chemotherapy-refractory endemic KS.human gammaherpesvirus-8 (HHV-8), now considered a necessary causal agent for the development of KS. The endemic KS subtype can follow an aggressive clinical course with ulcerative skin lesions with soft tissue invasion or even bone or visceral involvement. In the latter cases, a thorough imaging work-up and better follow-up schedules are warranted. As KS is a chronic disease, the therapeutic goal is to obtain sustainable remission in cutaneous and visceral lesions and a good quality of life. Watchful monitoring may be sufficient in localized cutaneous forms. Potential therapeutic modalities for symptomatic advanced KS include systemic chemotherapies, immunomodulators, immune checkpoint inhibitors, and antiangiogenic drugs.Kaposi’s sarcoma (KS) is a common neoplasm in Eastern and central Africa reflecting the spread of  Since ive tract , KS has ve tract ,4. Classve tract . Iatrogeve tract . Since tve tract .Recent discoveries have led to a better understanding of KS viral oncology and its clinical correlation, with a continuous objective to develop promising preventive and therapeutic targets. However, many questions remain to be answered to fully understand and address the multiple features of this disease.In this comprehensive review, our aim was to focus on endemic KS, and to summarize recent insights into epidemiological and geographical distribution, with a particular emphasis on clinical presentation and specific treatments.The Global Cancer Observatory 2020 data outline the age-standardized incidence rates for KS across continents irrespective of HIV status . With apWhile the latest data on KS in Africa reflect both HIV-related and unrelated diseases, it still mirrors to a certain extent the epidemiological pattern that existed before the advent of AIDS. The relative risk of KS in HIV seropositive individuals compared to HIV-seronegative individuals, remains a magnitude lower than would be expected in the United States ,10,11, hIn portions of Uganda, Tanzania, and the Democratic Republic of Congo, incidence rates of endemic KS, approached >9 per 1000 person years , whereasEndemic KS generally occurs at two age peaks: in the pediatric population, among children with a median age varying from 4 to 9 years ,19,20,21The epidemiology of KS in Sub-Saharan Africa and South Africa, an area with endemic HHV-8 and high HIV seroprevalence, is driven by both HIV-related KS and endemic KS.In the context of the HIV epidemic, a major increase occurred in the age-standardized KS incidence in both subtypes (HIV-related and endemic) by approximately 15%, in both female and male adults . NotablySince the HIV epidemic, endemic KS has been overshadowed by HIV-related KS and scarce data have been published on HIV-unrelated KS in Africa. Subsequently, high quality and accurate epidemiological data distinguishing between endemic forms of the disease according to HIV status are difficult to obtain.HHV-8 fulfills most of modern-day Koch’s postulates, linking this oncogenic virus to KS , as viraHHV-8 DNA sequences were identified, first in 1994, in all KS lesions from patients with AIDS . InitialThe recent focus on signaling pathways shows that HHV-8 immunoregulatory, growth-promoting and oncogenic genes can be expressed without the full execution of the lytic cycle ,61,62. TLong before the outbreak of HIV, KS was seen in children throughout Sub-Saharan Africa, suggesting a non-sexual pathway of transmission . The incAlthough HHV-8 infection is necessary for the development of KS lesions, it is not sufficient, and several factors could contribute to the maintenance of the disease .Environmental factors such as concomitant infections with parasitic agents may explain part of the geographical variation in HHV-8 prevalence . AccordiSince infection with HHV-8 has been consistently associated with KS lesions, several reports have examined the association between HHV-8 seroprevalence and low socioeconomic status markers in South Africa particularly among HIV negative individuals ,79. The Distinguishing KS cutaneous lesions from other benign or infectious vasoproliferative tumors involving the skin can be challenging. Bacillary angiomatosis illustrates one of the clinical misdiagnoses in the African region exhibiting both morphological and histological similarities . When reThe patch stage presents with patchy dilated leaky vessels mostly in the reticular dermis accompanied by a perivascular inflammatory infiltrate composed of lymphocytes and plasma cells. Plaque stage KS is characterized by diffuse dermal proliferation of spindle cells arranged in fascicles separating collagen bundles and forming irregular jagged-outlined vascular spaces. There is commonly an extravasation of red blood cells and hemosiderin deposits. Nodular lesions consist of a dense well-defined infiltrate of spindle cells along replacing the dermal collagen and forming pseudo-vascular spaces filled with red blood cells ,24,83. ICellular components of KS lesions typically involve spindle cells expressing endothelial markers such as CD34 and CD31 of vascular origin, as well as VEGF receptor 3, LYVE1, and podoplanin, highlighting its lymphatic endothelial cell origin . Recent Four clinically distinct categories of endemic KS have been identified but no staging classification has been validated to date: (1) a benign nodular form mimicking classic KS mostly occurring in young adults; (2) an aggressive form with local invasion of underlying soft tissue and bone; (3) a disseminated disease including florid mucocutaneous and visceral involvement, similar to HIV-associated KS before the advent of highly active antiretroviral therapy (HAART); and (4) a fulminant lymphadenopathic form with rapid lymph node and visceral disease particularly seen in children ,75. PatiEndemic KS occurs in young adults living in Sub-Saharan Africa, and typically manifests as localized mucocutaneous disease ,91. It pAs the cutaneous disease progresses, skin lesions may transition through the patch, plaque, nodular, infiltrative, or florid forms . NodularEndemic KS may less frequently involve extracutaneous sites including lymph nodes and visceral organs, particularly the respiratory and gastrointestinal tracts . In an oEndemic KS in children presents with clinical features different from those described in the adult subtype ,101,102.A distinct pattern of clinical presentation between HIV-negative endemic KS and HIV-related KS in the African setting was established by El-Mallawani et al. when comparing characteristics from both cohorts in the pediatric population of Malawi . Such diThe modified “Lilongwe Pediatric KS Staging Classification” , which hIn his initial description of KS, Moritz Kaposi reported cases with deep ulcerating lesions resulting in underlying periosteal damage and osteolytic lesions , referriPatients with bone involvement of KS may have localized pain , while oA review of 66 cases of KS with skeletal involvement conducted by Caponetti et al.  showed tMost bone lesions are osteolytic with cortical erosion or destruction secondary to KS lesions arising in the cortex or the periosteum. The conventional radiographic findings are variable, such as uniform rarefaction, cystic lesions , or bubbIn patients with aggressive localized infiltrative tumors, widespread disease, or bone pain, a thorough evaluation is warranted for detecting bone lesions. The detailed work-up should include CT scans for detecting cortical lytic changes and MR imaging for better identification of soft tissue masses and spongy bone involvement. Compared to conventional imaging studies, FDG-PET is mostly beneficial for whole-body staging and is also helpful for distinguishing visceral or skeletal involvement which can herald a worse prognosis . A retroIn low- and middle-income (LMI) settings, conventional radiography is readily available to help detect bone erosion or frank destruction as well as periosteal reaction. In cases where bone lesions go unrecognized and are difficult to detect on X-Ray, the use of a CT scan may provide better identification of osteolytic changes with cortical bone involvement ,116,117.Therapeutic management for patients with endemic KS must be tailored to the number of lesions, the extent of the disease and its functional consequences, and the presence of visceral involvement. Benajiba et al. . examineMinimally aggressive and localized forms of KS can respond favorably to local therapy but often tend to recur . The mai2 intravenous perfusion every three weeks) and alternatively paclitaxel (PTX)  [In widespread, progressive, and symptomatic forms, or with visceral involvement, systemic treatment is generally warranted . Based o-HIV KS) ,124,127.-HIV KS) . Due to -HIV KS) ,130,131,-HIV KS) . Similar-HIV KS) ,134,135.Alternatively, immune-modulating agents such as low-dose interferon alpha and its pegylated derivatives, used at three to five million units, represent an effective and well tolerated therapeutic modality for endemic KS in younger patients with normal cardiac function ,137.Recent preliminary data suggest the efficacy of anti-PD-1 in patients with classic and endemic KS. Major clinical and metabolic partial responses were obtained following nivolumab in two severe endemic KS patients with cutaneous, lymph node, muscular, and bone extension . A multiIn an open-label single-arm phase I/II study , pomalidWhile endemic KS patients usually respond favorably to systemic chemotherapy and/or radiation therapy, some patients may be refractory to these treatments. In cases of painful ulcerating rapid-growing KS tumors, with bone and/or muscular extension, and failure of standard and experimental treatments, limited amputation may be necessary .In 2020, there were an estimated 25,010 new cases of KS on the African continent and 13,066 deaths in the region . The gloGiven the rarity of pediatric KS, there are no therapeutic guidelines for pediatric KS. Most therapeutic modality options are extrapolated from published articles about KS in adults .Chemotherapy remains the cornerstone for treating children with endemic KS ,146. TheThe risk-stratification platform based on the “Lilongwe Classification” seeks to define therapeutic options predicting which patients may have a favorable prognosis with bleomycin and vincristine (BV) and those who will eventually require alternative chemotherapy ,149. AllThe World Health Organization (WHO) developed a Model List of Essential Medicines (EML) encouraging countries to prioritize specific anticancer drugs in national essential medicine lists and national reimbursable lists . The lisFurthermore, numerous public–private collaborations seek to supply and provide the quality cytotoxic medications required in treatment centers across Sub-Saharan Africa at affordable prices. Paclitaxel and doxorubicin appear among drugs prioritized for KS guidelines in countries such as Tanzania, Uganda, Zimbabwe, and Malawi .KS is one of the most common neoplasms in Sub-Saharan Africa and parallels the endemicity of HHV-8, the causative oncogenic organism. Other etiologic factors are specifically related to ethnic practices in African populations, such as HHV-8 salivary transmission.Endemic KS commonly presents with a monomelic variant consisting of a lymphedematous limb with large florid tumors locally infiltrating subjacent soft tissues and bones.However, although endemic KS is a leading malignancy in Sub-Saharan Africa, the diagnosis is challenging in resource-limited environments, as immunostaining techniques are not always available.KS remains a major public health concern in endemic regions as financial limitations represent a great challenge for conducting large therapeutic studies aiming to improve specific therapeutic management. To date, there are no curative therapeutic options, as HHV-8 is not eradicable. The purpose of tailored management is to adapt therapy to patient’s fitness and quality of life requirements. Treatment guidelines designating PLD and PTX as preferred first-line systemic agents are however, infrequently used because of their low availability and high cost. Further prospective trials are required to validate the use of PD1 blockade or pomalidomide in patients with endemic KS.


Vibrio vulnificus is an opportunistic pathogen that naturally inhabits sea water globally and is responsible for most vibriosis-related deaths. The consumption of V. vulnificus contaminated seafood and exposure of wounds to Vibrio can result in systemic infection, with increased risks of amputation and extremely high rates of mortality. However, the pathogenicity and virulence factors of V. vulnificus are not fully understood. The genomic characterization of V. vulnificus will be helpful to extend our understanding on V. vulnificus at a genomic level. In this manuscript, the genome of V. vulnificus strain MCCC 1A08743 isolated from contaminated prawns from Zhanjiang, China, was sequenced using Illumina HiSeq X Ten system and annotated through multiple databases. The strain MCCC 1A08743 genome included 4371 protein-coding genes and 117 RNA genes. Average nucleotide identity analysis and core genome phylogenetic analysis revealed that MCCC 1A08743 was most closely related to strains from clinical samples from the United States. Pathogenicity annotation of the MCCC 1A08743 genome, using Virulence Factor Database and Pathogen-Host Interactions database, predicted the pathogenicity of the strain, and this was confirmed using mice infection experiments, which indicated that V. vulnificus strain MCCC 1A08743 could infect C57BL/6J mice and cause liver lesions.This article has an associated First Person interview with the first author of the paper. Summary:Vibrio vulnificus strain MCCC 1A08743 was newly isolated, sequenced and tested for its pathogenicity in mice. Vibrio vulnificus is a gram-negative bacillus that is responsible for most of the world's vibriosis-related deaths. Vibrio vulnificus is usually found in warm, shallow, coastal salt water in temperate climates, and people generally become infected through either the consumption of V. vulnificus-contaminated seafood or the exposure of open wound. In the United States, V. vulnificus is the leading cause of seafood consumption-related deaths, and vibriosis is reported to cause ∼80,000 illnesses and ∼100 deaths each year (www.cdc.gov/vibrio/index.html). Some patients experience gastroenteritis, whereas others may suffer from severe forms of infection, such as sepsis, severe cellulitis, and necrotising fasciitis . The genome of MCCC 1A08743 has a size of 4,825,020 bp with a mean G+C content of 46.70%. A total of 4371 protein coding genes and 117 RNA genes were predicted, and the general features of strain MCCC 1A08743 are summarised –3.Table Table S1). The distribution of these genes into COGs (clusters of orthologous groups) functional categories was analysed, among which, signal transduction mechanisms ranked the largest category with 325 genes .The putative functions of most of the protein-coding genes were prognosticated through multiple databases, and the gene annotation of strain MCCC 1A08743 was summarised  of these strains, which suggested that global V. vulnificus is likely to be heterogeneous. Interestingly, strain MCCC 1A08743 was assigned to a specific branch with the strain NBRC15645. According to the NCBI database, NBRC15645 is equal to ATCC 27562, which was isolated from a clinical sample in the United States. Both strains were clustered with another two strains (ATCC 27562_2 and VN-0206), among which, ATCC 27562_2 represents the genome sequence of ATCC 27562 with contig level assembly completed in the year 2016, while VN-0206 was isolated from a German environmental sample . ANI varied from 95.04–100% among the 212 worldwide strains of V. vulnificus. Strain MCCC 1A08743 from China had a 95.30–99.99% ANI with the 211 other strains of V. vulnificus in the NCBI database. Strain MCCC 1A08743 harboured the highest identity (99.99%) with strain ATCC 27562_2, ATCC 27562_1, NBRC15645, and FDAARGOS_663, all of which were isolated from the United States clinical samples. ATCC 27562_1 represents the genome sequence of ATCC 27562 with complete level assembly completed in the year 2017. In addition, strain MCCC 1A08743 had the second highest identity (99.98%) with strain B2, isolated from human blood sample in China. Thus, the ANI analysis and phylogenetic analysis based on core genome reach the same conclusion, strain MCCC 1A08743 might have more similarities with clinically isolated V. vulnificus strains.ANI (average nucleotide identity) analysis was conducted with the full-length genome sequence of strain MCCC 1A08743 and 211 other V. vulnificus contribute to its pathogenicity have been reported, such as hemolysin, VvpE (Table S3). We further analysed the pathogenicity of strain MCCC 1A08743 through PHI base (the Pathogen-Host Interactions database), 501 genes were annotated, and the pathogen-host interaction phenotype classification was summarised in Several virulence factors of in, VvpE , RtxA1 tin, VvpE , the capin, VvpE , flagellin, VvpE . Thus, wV. vulnificus strains have been reported to cause systemic infection and even mortality in mice   A. This i 1.1758) . Thus, sV. vulnificus infections. One of the key functions of the liver is to remove blood-borne pathogens and to prevent the spread of bacteria to other organs and cause sepsis  and metabolic process (1548 genes).In recent years, outcomes . It is wV. vulnificus worldwide, we found that strain MCCC 1A08743 was most similar (99.99%) to strains derived from US clinical samples, which was consistent with the results of core genome comparison and phylogenetic analysis, and together, these results indicate that strain MCCC 1A08743 could harbour biological properties that are similar to those of strains from United States clinical samples. Although the V. vulnificus genes and processes leading to human diseases are not well understood, several canonical virulence factors of V. vulnificus either contributing to growth in serum, inflammation, immune evasion or invasion could be found in the genome of the strain MCCC 1A08743 . According to reports from the Centers for Disease Control and Prevention (CDC), patients with chronic liver disease or immunocompromising conditions are susceptible to V. vulnificus infection with a more severe phenotype  mouse model also showed a more severe phenotype than normal mouse model (data not shown), and we are exploring the mechanisms behind it.All the above information from genome analysis indicates the strain MCCC 1A08743 are infectious. Finally, an earchers . The Kaphenotype . In this disease . Anotherchanisms . HoweverV. vulnificus and may provide useful information for future epidemiological investigation and research of pathogenicity and virulence factors.In conclusion, this study improves the current genome-level understanding of Vibrio vulnificus strain MCCC 1A08743 was purchased from the Marine Culture Collection of China , with deposited number: MCCC 1A08743.A single colony of MCCC 1A08743 was inoculated into 5 ml brain heart infusion broth (BHI) and cultured at 28°C with vigorous shaking (180 rpm). Genomic DNA was isolated from 3 ml culture broth using an UltraPure™ DNA extraction kit  and the quality of the purified genomic DNA was evaluated using a NanoDrop 2000 spectrophotometer  and a Qubit 2.0 fluorometer . https://github.com/lh3/readfq) to remove adapter sequences and sequences with either low-quality bases or high N-percentages. SOAPdenovo 2.04 . About 1.1 gigabytes of clean data were obtained with over 200× coverage after using readfq  (https://rfam.xfam.org/), and then confirmed by the CMsearch program (Version 1.1rc4) with default parameters. Gene annotations were determined with the BLASTP program  and six databases including COG (Protein-coding genes were predicted using GeneMarkS version 4.17 (neMark/) . RepetitneMark/) , and TRFneMark/)  was usedneMark/) , and ribneMark/) . In addiding COG , GO . Core genes and specific genes of the 211 strains of V. vulnificus and MCCC 1A08743 were analysed using the CD-HIT (V4.6) fast clustering of similar protein sequence with default parameters . We determined the core genome cut-off through sensitivity analysis by maximizing the second derivative of the curve for the relationship between core genome genes number and the number of strains. The core genome profile trend represents the change in core genome to changes in the number of strains a gene must be found in to be defined as core   .V. vulnificus strains. Specifically, we chose a set of 2248 core genes through core genome analysis. The gene family clustering was based on the multiple sequence alignment results using MUSCLE software , 0.1 ml V. vulnificus CGMCC 1.1758 , or phosphate-buffered saline (PBS) with 0.01% (wt/vol) gelatine (negative control).Six- to eight-week-old specific pathogen-free male C57BL/6J mice weighing approximately 20 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . The mice were pre-treated with iron dextran at 1 h before treatment as previously described , and theV. vulnificus colony forming units (CFUs) of the mouse livers, infected mice were perfused with PBS, and then the livers were harvested, homogenised in PBS, diluted, and further cultured on BHI agar plates at 28°C for 24 h. For biosecurity, the infected mice were disposed by autoclaving at the end of the experiments. All animal experiments were approved by the Committee on Ethics of Medicine, Naval Medical University, PLA.The number of dead mice within 48 h and their survival time were recorded. Six mice were used for each dose, and the experiment was repeated twice. To measure the Vibrio vulnificus strain CGMCC 1.1758 was purchased from the Marine Culture Collection of China , with deposited number: CGMCC 1.1758. The original number of CGMCC 1.1758 is ATCC 27562.Specimens of liver were collected immediately after sacrifice and fixed in 4% paraformaldehyde solution (PFA). The fixed tissues were then embedded in paraffin and cut into thin sections (5–10 µm). Histological sections were routinely stained with Haematoxylin and Eosin.P-value <0.05 was considered significant.All statistical analyses were performed using GraphPad Prism 5.0 software. For comparisons, log-rank test was performed as indicated in figure legends to compare survival curves. A All animal experiments were approved by the Committee on Ethics of Medicine, Naval Medical University, PLA. This study was carried out in accordance with relevant institutional guidelines and regulations. All animal experiments were carried out in compliance with the ARRIVE guidelines.


Kallmann syndrome (KS) is an uncommon genetic disorder characterized by isolated congenital hypogonadotropic hypogonadism (CHH) and anosmia/hyposmia. KS originates from abnormal embryonic migration of olfactory axons and gonadotropin-releasing hormone (GnRH)-synthesizing neurons. It can be challenging to diagnose due to its heterogeneous clinical presentation and genes implied. Herein, we report a rare phenotype of KS in two sisters accompanied by a variety of nonreproductive disorders such as hypoparathyroidism, hypercortisolism, atrophy of the cerebellum, intellectual disability, and remarkably, ovarian dysgenesis. Additionally, both subjects present muscle weakness, exercise intolerance, marked hypotonia and seizures, being suspected, although not fully confirmed, mitochondrial encephalomyopathy. These cases illustrate the heterogeneous clinical presentation and the diagnostic difficulties often found in patients suffering from this condition. These clinical features have never been described before as associated with KS; therefore, we decided to report this novel KS phenotype. CHH is primarily caused by a GnRH deficiency, leading to low levels of gonadotrophins and sex steroids. In approximately 50% of the cases, CHH patients also present a defective sense of smell, which is then known as KS. Historically, KS and the normosomic CHH variant have been well differentiated. Nevertheless, recent studies suggest that from a clinical standpoint, these two entities should be considered two different expressions of the same pathology as they share common features and genetic etiopathogenesis , 2. TheyIn association with CHH, a wide spectrum of nonreproductive features and developmental abnormalities have been reported, although its prevalence is not well established yet. The most commonly described abnormalities include microphallus, cryptorchidism, synkinesis, hearing loss, renal anomalies, cleft lip/palate, and dental agenesis , 2. OvarThe diagnosis could be a formidable challenge due to its heterogeneous presentation. If it is recognized during the early stages, an appropriate approach and management of the disease can be made to minimize the long-term effects of hypogonadism .In recent years, genetic diagnosis has changed as new genes and inheritance patterns have been identified. Also, additional phenotypic features newly described could be used for the prioritization of genetic screening .Herein a report of two sisters affected by this condition is presented, highlighting phenotypic variability, diagnostic complexity, the importance of tailored treatment, and the need for future research.A highly exceptional case of KS in two sisters without substantial family background prompted this report. Permission from the family and patients was obtained prior to the publication of this article, as well as approval of the ethics committee (HCB/2020/1392).Case 1: The first patient is a female of 34 years old who was diagnosed with hypogonadotropic hypogonadism at the age of 16, and the suspicion of KS was confirmed at the age of 27. She presents the following nonreproductive features: hypoparathyroidism, hypercortisolism, intellectual disability, and interatrial communication. Due to her marked hypotonia, exercise intolerance, developmental delay, muscle weakness, and episodes of seizures, at the age of 20, a muscle biopsy was performed, and she was also diagnosed with mitochondrial encephalomyopathy due to the presence of COX-negative fibers with a deficit of complex I of the respiratory chain. Furthermore, because of her condition, at the age of 23, she was diagnosed with osteopenia. From an early age, she demonstrated neurodevelopmental disorder and intellectual disability. Multiple studies during her infancy and childhood were performed. A karyotype found no abnormalities (46 XX); sequencing of the mitochondrial DNA found no disease causing; and CAT and MRI of the brain described cerebellum atrophy, a microadenoma in the pituitary gland, and calcifications at the basal ganglia. At the age of 16, due to the absence of secondary sexual characters and the lack of growth spurt, further studies were made. Analysis showed indetectable sexual steroids and gonadotropins (< 0.10 UI/L) being compatible with congenital hypogonadotropic hypogonadism. At this point, ultrasonography and computerized tomography of the pelvis were performed, showing a rudimentary uterus and the presence of streak gonads. Laparoscopy with ovarian biopsies was performed, confirming gonadal dysgenesis: neither follicles nor germ cells were found in the biopsy. A growth hormone stimulation test revealed a partial deficit of the growth hormone (GH). Consequently, she was treated with GH replacement therapy that lasted for 6 months. After ending the GH replacement therapy, she began with estrogen therapy for 6 months. Since then, she has been under hormone replacement therapy with combined estrogen and progesterone. At a later stage, Kallmann syndrome was suspected as hyposmia was detected. A genetic test to detect KAL1 (ANOS1) and KAL2 mutations was performed, but both were negative. A comparative genomic hybridization was performed, resulting in a loss of heterozygosity in chromosome 13. Another brain MRI was performed and confirmed previously undetected aplasia in the olfactory bulbs Fig. .Fig. 1BrCase 2: The second patient, a sister of case 1, presented a developmental course, diagnosis process, and diagnostic studies almost analogous to those previously described. She is a female of 29 years old who was diagnosed with CHH at the age of 14, and the diagnosis of KS was established at the age of 22. She presents the following nonreproductive features: hypoparathyroidism, high cortisol levels, intellectual disability, mitral valve prolapse, and subclinical hypothyroidism. She also presents muscle weakness, exercise intolerance, and marked hypotonia. Her muscle biopsy was considered normal, but a diagnosis of mitochondrial encephalomyopathy was made, assuming it was a false negative.She was diagnosed with spina bifida at birth and also presented neurodevelopmental disorders during early life, resembling patient 1. The same studies carried out for patient 1 were performed during her early life: karyotype and mitochondrial DNA sequencing, both with no abnormalities. Computerized tomography and MRI of the brain also described cerebellum atrophy and calcifications at the basal ganglia. Like her sister, she exhibited linear growth but not growth spurt during adolescence. At the age of 14, she was detected to have a deficiency in growth hormone and CHH, after which she began GH treatment. A year later, at 15, estrogens were added to the GH therapy. Since 16 until now, she has been receiving hormone therapy with estrogen and progesterone. Just like occurred to patient 1, image studies suggested rudimentary uterus and streak ovaries; thus, a laparoscopy and an ovarian biopsy were performed, and gonadal dysgenesis was confirmed. Further on, anosmia was also detected, and KS was suspected. In an analogous manner to that of her sister, no mutations were found in KAL1 and KAL2, but it was confirmed on her a loss of heterozygosity of chromosome 13. Furthermore, at the age of 17, she was diagnosed with osteopenia. Brain MRI revealed a mega cisterna magna, atrophy of cerebellar vermis, small pituitary gland, and aplasia in the olfactory bulbs and tract  gene. The comparative genomic hybridization performed in both patients did not report which part of chromosome 13 was involved in the loss of heterozygosity, hindering the possibility of finding a candidate gene involved in the etiology. Conversely, chromosome 13 has not been related to any genetic forms of HH or KS specifically . The autAlthough genetics remain to be fully elucidated in KS, recent research has led to the identification of more than 60 genes that might be implicated in approximately 50% of the cases , 6, 9. CA proper genotype–phenotype correlation might aid in addressing genetic testing and to personalizing genetic counseling and screening . Some spMitochondrial myopathies are a group of heterogeneous disorders caused by an impairment in the oxidative phosphorylation pathways of the respiratory chain. The diagnosis is usually suspected by a detailed history and physical examination and can be confirmed by molecular genetic studies, muscle biopsy, and additional tests such as neuroimaging and electromyography , 14. In The most exceptional finding in these patients is the fact that gonadal dysgenesis was demonstrated at the ovarian biopsies of both. To our knowledge, there is only one case report in the literature in which ovarian dysgenesis has been manifested concurrently with KS . GonadalIt is also noteworthy that some studies have described impairment of smell in patients with hypergonadotropic hypogonadism, more specifically, in patients with Turner syndrome . This coAppropriate management is crucial to diminish health consequences . The lacThese two cases highlight the genetic and phenotypic heterogeneity of KS and spotlights that there is still a broad array of unanswered questions. We contemplate that further research is needed to determine the molecular basis correlating CHH, intellectual disability, gonadal dysgenesis, and mitochondrial encephalomyopathy underlying these two cases as it has not been reported in the literature before. Specifically, we highly encourage to deeply study possible aberrations in 13q33-q34 in these patients and their parents in order to eventually shed light on its inheritance pattern.


About 2 years ago when the journal gave us the opportunity to launch this Research Topic, the three of us were excited by the rapid growth of cutting-edge imaging approaches now illuminating the architecture and molecular organization of synapses. Since its launch, we received enthusiastic responses from many authors and in the end, we published 15 articles in this volume.These articles, including original research, reviews, and opinion, present a snapshot of several key active areas of ongoing work. They fall roughly into three areas: technical advances in single-molecule imaging and analysis, dynamics of molecular organization within the synapse, and advances in electron microscopy that are propelling new insights.Unterauer and Jungmann provided an updated review of DNA PAINT technology which provides single-nanometer resolution with straightforward routes to molecular counting along with highly multiplexed imaging. A great advantage of the approach is that with a single labeling step, antibodies conjugated with short DNA oligonucleotides can label multiple targets that are subsequently imaged by sequential exchange of fluorophore-labeled complementary oligonucleotides, avoiding chromatic aberration. Narayanasamy et al. then further expanded the use of DNA-PAINT by demonstrating multiplexed imaging of multiple pre- and post-synaptic proteins in brain slices, demonstrating the viability of this important approach for analyzing synaptic nanostructure in vivo. In a Perspective, Specht argued that single-molecule imaging provides a powerful approach to determining protein copy number, providing both high throughput and high subcellular spatial resolution to interpret such counts. Copy number of key postsynaptic density (PSD) proteins have been studied using calibrated fluorescent light microscopy  receptor subunits by genetic code expansion with unnatural amino acids combined with bio-orthogonal click-chemistry labeling with tetrazine dyes. Gagliano et al. provided a comprehensive review of single molecular tracking and super-resolution imaging of synaptic proteins in 3D enabled by new light sheet illumination approaches. Sneve and Piatkevich provided an overview of new insights that may arise from using expansion microscopy to map synapses in neuronal circuits. These contributions highlight the enormous potential of combining novel labeling approaches with novel microscopy techniques.Several new advances within this arena were also introduced in this Topic. Piao and Sigrist provided a detailed review of work from Drosophila which shows how the active zone protein (M)Unc13 defines the behavior of individual release sites of the active zone. More generally, the work illustrates that isoform-specific components of the vesicle release machinery may drive functional presynaptic heterogeneity, revealing principles likely at play in the mammalian brain as well. In new research, Maschi et al. imaged release of individual vesicles , new and improved sample preparation techniques and advances in image processing software development are all powering new discoveries.Watanabe et al. reported their development of sets of image analysis programs for aiding quantitative morphometric analysis  of subcellular organelles such as vesicles, endosomes in synaptic terminals, this would be especially useful for large bulk of serial section EM images where manually analysis might be too daunting and unproductive; they also introduce a scheme to address sampling biases in image analysis, a much needed feature to have.Here we collected three EM related papers as vignettes of this blooming field. To further dissect structural basis of synaptic transmission, information of subsynaptic structures/organelles now often need to be quantified in specifically designed experiments. Petralia et al. provided a review of a class of underappreciated structures called invaginations, small outward projections from one cell membrane to another in various synaptic junctions, which is often too small to be seen by light microscopy and was often misidentified by traditional thin section EM. Invaginations appear to be important for further understanding processes in synaptic development, maintenance, and plasticity. Now, focused ion beam-scanning electron microscopy (FIB-SEM), one of the new 3D EM or volume EM methods, makes tracing and detailed analysis of these invaginating structures possible.3D volume EM uses fixed, heavy metal-stained, and plastic-embedded brain tissues imaged by serial block-face scanning EM (SBEM), requiring either mechanical serial sectioning . The organization of these active zone materials and its structural model was based on 3D reconstructions from EM tomography of fixed or freeze-substituted frog NMJ. A molecular level structural model detailing vesicle fusion processes at the presynaptic terminal of the NMJ active zone might have broader implications for understanding the molecular basis of synaptic transmission in other types of synapses in general. Super-resolution light microscopy is now revealing the molecular organization of active zone proteins at NMJ (Badawi and Nishimune, Finally, Transmission EM tomography of synapses has allowed the creation of 3D reconstructions to delineate organization of subsynaptic organelles, key synaptic proteins, and macromolecular complexes at pre and postsynaptic terminals in fixed or high pressure frozen and freeze-substituted neuronal culture, brain slice culture or nerve tissue (Harlow et al., Together, the articles of this Research Topic highlight that synaptic nanoarchitecture is a blossoming field helping to grow and propagate important technical advances and unearthing new insight to synaptic function. We close this volume of the topic with hope that we will come back to this lively garden of ours in due time to welcome more papers that highlight exciting new progress and development in this vibrant field. We look forward to seeing you then!XC, TK, and TB conceived the main ideas and wrote the manuscript. All authors contributed to the article and approved the submitted version.XC is supported by the NINDS intramural program. TB is supported by R37MH080046. TK is supported by grants of the Baden-Württemberg Stiftung.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.


This study was designed to investigate the regulatory effect of hyaluronic acid (HA)—coating silk fibroin (SF) nanofibers during epithelialization of urinary tract for urethral regeneration. The obtained electrospun biomimetic tubular HA‐SF nanofiber scaffold is composed of a dense inner layer and a porous outer layer in order to mimic adhesion and cavernous layers of the native tissue, respectively. A thin layer of HA‐gel coating was fixed in the inner wall to provide SF nanofibers with a dense and smooth surface nano‐topography and higher hydrophilicity. Compared with pure SF nanofibers, HA‐SF nanofibers significantly promoted the adhesion, growth, and proliferation of primary urothelial cells, and up‐regulate the expression of uroplakin‐3 . Using the New Zealand male rabbit urethral injury model, the scaffold composed of tubular HA‐SF nanofibers could recruit lumen and myoepithelial cells from the adjacent area of the host, rapidly reconstructing the urothelial barrier in the wound area in order to keep the urinary tract unobstructed, thereby promoting luminal epithelialization, smooth muscle bundle structural remodeling, and capillary formation. Overall, the synergistic effects of nano‐topography and biophysical cues in a biomimetic scaffold design for effective endogenous regeneration. ATR‐FTIRFourier transform infrared spectra with attenuated total reflection headDAPIdiamidino‐2‐phenylindoleDMEMDulbecco's modified Eagle's mediumECMextracellular matrixEDC1‐ethyl‐3‐[3‐dimethylaminopropyl] carbodiimide hydrochlorideFBSfetal bovine serumHAhyaluronic acidHFIPhexafluoroisopropanolIgGimmunoglobulin‐GK5cytokeratin‐5P/Spenicillin/streptomycinPBSphosphate buffered salineRTroom temperatureSEMscanning electron microscopySFsilk fibroinSMCsmooth muscle cellTCPStissue culture polystyreneTGAthermo‐gravimetric analysisUCurothelial cellsVVGVerhoeff Van‐GiesonXPSX‐ray photoelectron spectroscopyα‐SMAα‐smooth muscle actin1Urethral trauma is a frequent and costly injury. It may be caused by a multitude of factors, including physical injury, inflammation, ischemic stenosis, congenital defects, or malignant tumor.In situ tissue‐engineering using degradable biomaterials can utilize the regeneration potential of human body through reasonable scaffold design  and reproduce natural tissue regeneration.Urothelium is a stratified transitional epithelium derived from the endoderm, extending from the renal pelvis to the proximal urethra, serving as a key barrier between the blood and urine.Among the potential migration enhancements for epithelial cells, hyaluronic acid (HA), a critical element of extracellular matrix (ECM),Silk fibroin (SF) is a natural protein with excellent mechanical properties, good cell compatibility, controllable degradation, and versatile process‐ability in different material formats.22.1Using electrospinning, the inner wall surface nano‐topography of tubular SF and HA‐SF nanofibers present a smooth and porous nanofiber network, with the average diameters in the range of about (253 ± 15) nm and (222 ± 10) nm, respectively . To increase architecture stability, tubular SF and HA‐SF nanofibers were treated with EDC/ethanol solution. The nano‐topography of tubular SF and HA‐SF inner surface changed significantly after treatment Figure . The surHA is a highly hydrophilic biopolymer, so it is reasonable to expect that the SF nanofibers coated by HA have high wettability, which can be determined by water contact angle measurement. As shown in Figure −1, consistent with that of amide I, amide II, and amide III, respectively. Compared with SF nanofiber film, the ATR‐FTIR spectra of HA‐SF nanofiber show obvious additional absorption peaks at 1646, 1556, and 1287 cm−1. The band shifts of amide I and II were observed. Other studies have found similar band shifts.d10 of HA‐SF nanofiber is 366.32°C, which is between that of HA and SF nanofibers , further confirms the existence of HA‐coating.The chemical distribution of HA‐SF nanofibers was determined by ATR‐FTIR Figure . In the Both SF and HA‐SF nanofibers were tested in tensile mode to generate stress–strain curves and derive tensile properties . The Young's modulus and ultimate tensile strength of HA‐SF nanofibers in dry state are (0.83 ± 0.4) MPa and (1.7 ± 0.4) MPa, respectively, which are close to those of SF nanofibers. However, the elongation at break of HA‐SF is (387 ± 17)%, which is slightly lower than that of SF nanofibers (407 ± 20)%. These tests showed that HA‐SF and SF nanofibers are soft but tough substrate. Moreover, the Young's modulus of HA‐SF and SF nanofibers from AFM indentation test in wet state is (0.82 ± 0.16) and (1.2 ± 0.15) MPa, respectively , which is very close to that of HA‐SF and SF nanofibers in dry state. The Young's modulus of HA‐SF and SF nanofibers is higher than that of natural urethra (~0.3 MPa).2.2We studied the primary UCs growth behavior on the surface of SF and HA‐SF nanofibers. Figure Meanwhile, uropakin‐3 was used to visualize urothelial plaque in UC to evaluate waterproof and anti‐injury functions. Figure To study the effects of SF and HA‐SF nanofibers on the proliferation of UCs, we performed immunofluorescence staining of Ki67, a recognized proliferation marker located in the nucleus. In Figure 2.3Tubular SF and HA‐SF nanofibers were both applied to a New Zealand rabbit model of iatrogenic urethral trauma  in order to determine their effect on the process of urethral reconstruction in vivo. We transected New Zealand rabbit urethras and bridged the resulting urethras gaps with tubular SF and HA‐SF nanofibers, respectively Figure . X‐ray uVerhoeff Van‐Gieson (VVG) and Sirius red staining were performed to observe the urothelial regeneration at 14 weeks after operation Figure . VVG and2.4p > 0.05), and significantly higher than that (12 ± 1.2)% of SF graft group. These results suggest that HA‐SF grafts can induce structural remodeling of smooth muscle cells, rather than recruitment of single cells. As the outer layer of tubular HA‐SF nanofibers is mainly SF nanofibers, its biophysical clues and surface properties are exactly the same as those of pure SF nanofibers. Therefore, we speculate that the urinary epithelium on the inner wall of tubular HA‐SF graft may determine the tissue remodeling pattern of SMC on its outer wall. The formation of vascular network in regenerated tissue is the key to tissue integration.Next, Masson's trichrome staining was performed to evaluate the regeneration effect of the smooth muscle bundle and collagen nanofibers in the regenerated urethra Figure . At 14 w3In this study, we developed a biomimetic tubular nanofiber scaffold that promotes urethral functional recovery by recruiting healing‐associated endogenous UCs to reconstruct the urinary tract epithelial barrier. We found that by precisely controlling the biophysical and biochemical properties of biomaterial scaffolds, endogenous urethral cells can be guided to the defect site to achieve tissue regeneration. This method harnesses the regenerative potential of the human body and eliminates the need for ex vivo cell manipulation.In addition to the limited number of endogenous stem cells and progenitor cells in cartilage, heart, and nerve tissues, in situ tissue engineering provides temporary solutions and alternatives for most of the tissue regeneration.Herein, urethral graft based on HA and SF with fibrous structure was successfully prepared by sequential electrospinning. The biomimetic nano‐topography on the inner wall surface of tubular HA‐SF nanofibers is enhanced by crosslinking process. By adjusting the size of the receiving device (mold), it is convenient to realize the specific needs of patients for size and diameter. The tubular scaffold was composed of a dense inner layer and a porous outer layer. Its structure simulated the submucosa and cavernous layer of native urethral tissue, respectively. The nano‐topography of the inner wall surface of the crosslinked HA‐SF nanofibers mimics the native urethral epithelium. The main advantage of this structural design is that it successfully combines the protein with polysaccharide, which mimic the key physiological features of ECM.One of the advantages of biomimetic tubular HA‐SF nanofiber scaffold is the exploitation of regenerative potential of organism, controlling cell function to realize in situ tissue reconstruction. Moreover, the use of endogenous UCs for in situ urethral regeneration can also reduce immune rejection of transplanted (exogenous) cells, one of the main risks associated with cell therapy.44.1HA  was obtained from Meilunbio®. SF solution  was obtained from Sigma. Hexafluoroisopropanol  was purchased from Aladdin. Dulbecco's modified Eagle's medium , fetal bovine serum , penicillin/streptomycin , trypsin , phosphate‐buffered saline , Rhodamine‐ and Alexa Fluor‐488 conjugated phalloidin (R415 and A12379), DAPI (62248) were purchased from Gibco. Primary antibodies to cytokeratin‐5 , uroplakin‐3 (ab187646), α‐smooth muscle actin , CD31 (ab24590), and Ki67 (ab15580), and secondary antibodies for fluorescence staining were purchased from Abcam.4.2The HA solution was prepared by dissolving 200 mg HA in 10 ml HFIP and stirring until a uniform solution was obtained. A syringe with 10 ml HA solution was fixed on the precision injection pump. The flow rate of the electrospinning device was set at 1.5 ml/h, the distance between the electrospinning nozzle and the receiver (stainless steel with an outer diameter of ~2.7 mm or stainless steel rod of 10 mm) was set at 15 cm, while voltage was set at 12.3 kV. The speed of receiver was set at 1000 rpm, the temperature and humidity were 25°C and 45%, respectively. Electrospinning stopped after 2 or 6 h. A second syringe was loaded with 10 ml of fully mixed SF solution (50 mg/ml) and fixed to the precision injection pump. After connecting the electrospinning nozzle, the flow rate of the electrospinning device was set to 2.8 ml/h. The purpose of using a receiving device with 10 mm stainless steel rod was to obtain SF fiber film with HA‐coating on inner surface, while stainless steel with diameter of 2.7 mm was used to obtain tubular HA‐SF scaffold with inner diameter of 2.9 mm. HA‐coating SF nanofiber scaffold with an inner diameter of 2.7 mm and an outer diameter of 3.5 mm was obtained after 48 h electrospinning. The electrospinning setup of pure SF nanofiber film and tubular nanofiber scaffold in this study is consistent with the HA‐SF electrospinning procedure described above, but without the second HA electrospinning step.The as‐prepared nanofiber films and tubular scaffolds were dried in a vacuum drying oven for 48 h to remove HFIP. The samples were then classified and immersed in the ethanol solution containing 50 mM 1‐ethyl‐3‐[3‐dimethylaminopropyl] carbodiimide hydrochloride (EDC) for 24 h, respectively. After crosslinking, the nanofiber samples were placed in a vacuum drying oven to remove organic solvents.4.3−1 and scanning resolution of 2 cm−1.2. The mechanical properties of electrospun nanofiber samples were measured and calculated by a tensile testing machine equipped with a 50 N load cell and a built‐in software . The samples were cut into 1.1 × 6 × 0.5 cm2 and stretched at a constant rate of 5 mm/min.The surface morphology and the diameter of the nanofibers were observed by SEM  at an accelerating voltage of 5 kV.4.42 humidified incubator. After three passages, cells were seeded at a concentration of 106 cells/cm2 on nanofibers and the medium was changed every day for the next 5 days. Prior to cell seeding, the nanofibers (diameter: φ = 24 mm) on TCPS of 6‐well plate were sterilized by ionizing radiation at 10 kGy from 60Co for 15 min.The UCs of New Zealand rabbits were isolated using previously described methods,At a predetermined time point of 96 h postseeding, the cell morphologies on the nanofibers were studied. The cell seeding nanofiber samples were washed twice with PBS to remove floating cells, and then fixed with 4% paraformaldehyde for 15 min at room temperature (RT). The fixed samples were washed three times with PBS, then dehydrated through an ethanol gradient , and vacuum freeze‐dried over‐night, and conductive coating was implemented before SEM analysis, used to observe the status and morphology of cell growth on the nanofibers.After 96 h of co‐culturing, the cell scaffolds were fixed according to the above steps, embedded with optimum cutting temperature compound  frozen embedding agent  at −20°C, and finally sliced (20 μm) using constant cold box slicer at −22°C. Then the samples were stained with H&E staining kit (ab245880) according to the manufacturer instructions. The samples were visualized by pannoramic desktop microscope, with the representative areas were captured by Caseviewer software (version 2.3).4.5https://imagej.net/citing) considering 10 random fields per sample and n = 4 samples per group.After culturing for 24 and 120 h, the fixed cells seeding the nanofiber samples were permeabilized using 0.1% Triton X‐100 for 15 min and blocked with 2% serum albumin  for 30 min at RT. Then, the samples were incubated overnight at 4°C with primary antibodies uroplakin‐3 or Ki67 diluted 1:50 with PBS. Subsequently, after washing twice with PBS, the samples were incubated with Alexa Fluor‐488, or Cy3 conjugated anti‐mouse or anti‐rabbit immunoglobulin‐G (IgG) secondary antibodies diluted 1:400 with PBS for 30 min at RT. The F‐actin filaments of UCs were stained with Rhodamine‐ or Alexa Fluor‐488 conjugated phalloidin diluted 1:1000 with PBS for 30 min at RT, after washing twice with PBS the cell nuclei were stained with DAPI diluted 1:2000 with PBS for 10 min at RT. After washing three times with PBS, images were acquired using a Leica TCS SP5 CLSM. Uroplakin‐3+ and Ki67+ areas were shown as a percentage of the defined region area calculated using the ImageJ program  was used to estimate the cell proliferation. We considered the cells seeding on six‐well plate TCPS as the positive control group. The cells were cultured with the same medium for 24 and 120 h, respectively. After medium replacement, the cells were washed three times with warm PBS, then filled with 100 μl CCK‐8 reagent and 900 μl serum‐free DMEM subsequently. After co‐culturing for 4 h, the supernatant was transferred into 96‐well plates and the absorption was measured with a microplate reader  at 450 nm. Four parallel tests were performed for each sample.4.6All experimental procedures involving animals in this study were conducted under Institutional Guidelines for Animal Care and approved by the Animal Ethics Committee of Guangzhou Medical University . Male New Zealand rabbits aged 14 weeks were divided into two research groups: SF and HA‐SF scaffold graft group. Each animal received one graft, with nine animals in each group. The positive control was healthy urethral tissue, which was obtained from the 2 cm long urethral tissue cut during the manufacture of urethral trauma. As previously described,2) was isolated from the above‐mentioned healthy urethral tissue, while decellularized native urethral epithelial tissue was obtained by digestion with 0.2% collagenase IV  at 37°C for 2 h .Urothelial epithelial tissue , and the urethral tissue was dissected and separated. The retrieved graft samples were washed with normal saline to remove blood stains, fixed with 4% paraformaldehyde at RT for 24 h, dehydrated with gradient ethanol, embedded in paraffin, and cut into tissue sections (5 μm) were cut.At 6 weeks, and 14 weeks after urine flow rate experiment, the research animals were randomly sacrificed with an overdose of sodium pentobarbital , followed by the immunofluorescence staining protocol described in Section 4.5. The average K5, Uroplakin‐3, CD31, or α‐SMA positive expression area was measured on 10 random fields of each slide  with ImageJ software.4.8p < 0.05). Unless otherwise stated, data and error bars are reported as mean ± standard deviation.All experimental data were analyzed using GraphPad Prism  program. Where appropriate, a one‐way or two‐way analysis of variance (ANOVA) was performed to determine the significant difference with the Tukey post hoc test ; funding acquisition (supporting); investigation ; project administration ; resources (lead); writing – original draft (lead). Massimiliano Galluzzi: Investigation (supporting); writing – review and editing (lead). Fuming Deng: Investigation ; methodology . Zhang Zhao: Investigation ; methodology . Ming Fu: Investigation ; methodology . Liang Su: Investigation . Weitang Sun: Investigation . Wei Jia: Investigation . Huiming Xia: Conceptualization ; funding acquisition (lead); project administration (lead); supervision (lead).All authors declared no potential conflicts of interest.AppendixS1: Supporting informationClick here for additional data file.


A novel highly water-soluble 1,8-naphthalimide with pH and viscosity-sensing fluorescence was synthesized and investigated. The synthesized compound was designed as a molecular device in which a molecular rotor and molecular “off-on” switcher were integrated. In order to obtain a TICT driven molecular motion at C-4 position of the 1,8-naphthalimide fluorophore, a 4-methylpiperazinyl fragment was introduced. The molecular motion was confirmed after photophysical investigation in solvents with different viscosity; furthermore, the fluorescence-sensing properties of the examined compound were investigated in 100% aqueous medium and it was found that it could be used as an efficient fluorescent probe for pH. Due to the non-emissive deexcitation nature of the TICT fluorophore, the novel system showed low yellow–green emission, which represented “power-on”/“rotor-on” state. The protonation of the methylpiperazine amine destabilized the TICT process, which was accompanied by fluorescence enhancement indicating a “power-on”/“rotor-off” state of the system. The results obtained clearly illustrated the great potential of the synthesized compound to serve as pH- and viscosity-sensing material in aqueous solution. Due to the rapid progress in the fields of chemosensing materials and nanotechnology, the design and synthesis of molecular switches have received considerable attention in recent years ,2,3,4. AThe lack of water-solubility is the main disadvantage of fluorescent probes, which seriously restricts their usage. Currently, chemical analysis in water solutions, in the absence of organic solvents, was preferable due to the use of environmentally friendly media with lower cytotoxicity and higher biocompatibility, which is, especially, suitable for bioimagining purposes; therefore, the design and synthesis of highly water-soluble fluorescent probes has increasingly attracted considerable interest ,27,28,293) was designed as a fluorescence-sensing TICT rotor based on a 4-piperazinyl-1,8-naphthalimide architecture. The 1,8-naphthalimide fluorophore was chosen as a fluorogenic unit due to its bright fluorescence, large stokes shift, and high photo and chemical stabilities +.To a solution of In conclusion, a novel highly water-soluble 1,8-naphthalimide with pH- and viscosity-sensing fluorescence was designed using TICT molecular motion. The photophysical investigation of the synthesized compound was performed in an aqueous solution and in solvents with different viscosities. The results obtained revealed its potential to serve as a fluorescent probe for rapid detection of pH and viscosity, which was attributed to the destabilization of the TICT excited state in acid media and hindered rotation at high viscosity. The results presented here could be seen as a contribution to the development of the applied sensory chemistry using environmentally friendly aqueous media.