Mitochondria have beneficial effects on cells by producing ATP and contributing to various biosynthetic procedures. On the other hand, dysfunctional mitochondria have detrimental effects on cells by inducing cellular damage, inflammation, and causing apoptosis in response to various stimuli. Therefore, a series of mitochondrial quality control pathways are required for the physiological state of cells to be maintained. Recent research has provided solid evidence to support that mitochondria are ejected from cells for transcellular degradation or transferred to other cells as metabolic support or regulatory messengers. In this review, we summarize the current understanding of the regulation of mitochondrial transmigration across the plasma membranes and discuss the functional significance of this unexpected phenomenon, with an additional focus on the impact on the pathogenesis of cardiovascular diseases. We also provide some perspective concerning the unrevealed mechanisms underlying mitochondrial ejection as well as existing problems and challenges concerning the therapeutic application of mitochondrial ejection. Mitochondria are double membrane-bound α-proteobacterium-derived organelles that contain extrachromosomal deoxyribonucleic acid (DNA), termed mitochondrial DNA (mtDNA) . Most ofRecent investigations have revealed that unexpected mechanisms are involved in maintaining mitochondrial quality in cells by transporting mitochondria between the inside and outside of cells and across plasma membranes. For example, it has been shown that nerve cells, CMs, and adipocytes are able to eject damaged mitochondria from their cytosol ,15,16. MIn 2014, Davis et al. detected transcellular mitochondrial degradation from retinal ganglion cell axons to astrocytes under baseline conditions in vivo . Using tCaenorhabditis elegans (C. elegans) model [C. elegans eject “exophers”, membranous vesicles of 2–15 μm in diameter which contain mitochondria and other organelles or proteins. The production of exophers is provoked by various stresses, the suppression of autophagy or UPS activities, and mitochondrial damage. Recently, Nicolás-Ávila et al. revealed a transcellular mitochondrial degradation pathway between CMs and macrophages in healthy myocardium [+ exophers for ejection to extracellular spaces in baseline, and isoproterenol-induced cardiac stress promoted the ejection of exophers  model . The neuocardium . They foexophers A. Releasexophers A. In accRosina et al. revealed that the transcellular degradation of damaged mitochondria between brown adipocytes and brown adipose tissue (BAT)-resident macrophages was mediated through extracellular vesicles (EVs) . Under cMitocytosis is a migrasome-mediated mitochondrial disposal process that is regarded as a crucial mitochondrial quality control system in migrating cells . When ceThe intercellular transport of mitochondria was first reported by Koyanagi et al. in 2005 . A subseIt has been shown that MSCs alleviated airway epithelial cells from lipopolysaccharide (LPS)-induced acute lung injury  and asthRecent investigations revealed that the intercellular transport of mitochondria could also be seen between mature cells. Astrocytes transport mitochondria to neurons to protect the neuronal function in response to transient focal cerebral ischemia in mice  and cispThe intercellular transport of mitochondria is also involved in the genesis, survival, metastatic transformation, and drug resistance of malignant tumors . Thus faInterferon (IFN) and tumor necrosis factor (TNF) responses in endothelial cells. On the other hand, the loss of mitochondrial respiratory capacity, the presence of pyruvate, and MitoTEMPO, a mitochondrial reactive oxygen species scavenger, dramatically reduced the inflammatory response. Hough et al. reported that mitochondria-containing EVs could be seen in the bronchoalveolar lavage fluid of both healthy volunteers and asthmatics [It is widely recognized that mitochondria exert an immunomodulatory function through various mechanisms. The metabolic activities and metabolites of mitochondria are related to the regulation of immune activities of various immune cells. Similarly, mtDNA is closely associated with immunoreaction as a damage-associated molecular pattern (DAMP). Recently, immunoregulation mediated by “intercellular mitochondrial transfer” has received attention. Boudreau et al. substantiated the existence of functional mitochondria in EVs released by platelets, which was found in platelet concentrates used for transfusion . They althmatics . Althoug+CCR6+CD45RO+ T helper 17 (Th17) cells cocultured with MSCs, transported mitochondria from MSCs caused interconversion of Th17 cells into T regulatory cells, which resulted in the significant impairment of IL-17 production from Th17 cells [+ T cells, thereby promoting the activation of T-cells or differentiation into regulatory T cells. Indeed, the transplantation of MSC-derived mitochondria-treated CD4+ T cells significantly reduced the organ damage caused by inflammation and improved the survival rate in murine graft-versus-host disease models [A growing body of evidence suggests that mitochondrial transport by MSCs plays an important role in the regulation of immune responses. In an animal model of acute respiratory distress syndrome (ARDS), MSCs assisted the removal of bacteria by transferring mitochondria to macrophages via tunneling nanotubes (TNTs)  or mitoc17 cells . A subsee models . In addie models .Although numerous studies have revealed several routes of mitochondrial release/transfer, including TNTs and MVs, the specific molecular mechanisms remain to be elucidated. In this article, we have summarized the current understanding of the molecular mechanisms involved in the mitochondrial release and/or transfer from cells .TNTs, a unique intercellular membrane structure, were first reported in rat pheochromocytoma PC12 cells . TNTs haEV is another important transporter of mitochondria B. EVs arIn addition to participating in TNT-mediated mitochondrial transfer, CD38 is involved in the extracellular release of mitochondrial particles by astrocytes . As mentMitochondria can be ejected from cells or transferred to other cells through various other pathways. Activated platelets release functional mitochondria through the actin-dependent pathway, which is independent of the microtubule-dependent pathway. This process is considered to be a damage-associated mechanism . LPS-treIn summary, we reviewed the functional significance of mitochondrial transmigration across the plasma membranes in various cells. We also summarized the trends in investigations of such phenomena as novel therapeutic targets of various diseases, including heart failure. In recent years, mitochondrial supplemental therapy—based on mitochondrial transfer—has been proven to improve the function of various damaged tissues and organs. Previous studies have verified the potential efficacy of MSC transplantation-mediated mitochondrial supplemental therapy in animal experiments ,80. WithIncreasing lines of evidence suggest that the ejection of damaged mitochondria plays an important role in maintaining the quality of intracellular mitochondria. However, our understanding of the molecular mechanisms involved in the regulation of such a process remains limited. Recent studies have shown that MDVs are involved in the extracellular ejection of damaged mitochondria via EVs ,24. Howe


The technological advances in cochlear implants and processing strategies have enabled subjects affected by severe to profound hearing loss to hear sounds and recognize speech in various different degrees. The variability of hearing outcomes in subjects with post-lingual deafness has been significant and cochlear implant indications have been extended to include an ever larger population.This paper aims to look into the groups of post-lingual deafness patients to find where cochlear implants have yielded better outcomes than conventional hearing aids.Review the literature available on databases SciELO, Cochrane, MEDLINE, and LILACS-BIREME. The publications selected for review were rated as A or B on evidence strength on the day of the review. Their authors analyzed and compared hearing aids and cochlear implants in populations of post-lingually deaf patients. Study Design: Systematic review.Eleven out of the 2,169 papers searched were found to be pertinent to the topic and were rated B for evidence strength. Six studies were prospective cohort trials, four were cross-sectional studies and one was a clinical trial.The assessment done on the benefits yielded by post-lingually deaf subjects from cochlear implants showed that they are effective and provide for better results than conventional hearing aids. The technological advances in cochlear implants and processing strategies have enabled subjects affected by severe to profound hearing loss to hear sounds and recognize speech in various different degreesSeveral studies have been published within the last few years comparing the hearing outcomes obtained by severe and profound hearing loss patients using conventional hearing aids or cochlear implantscochlear implants/cochlear implantation; hearing aid; therapy; prognosis; comparative study and the corresponding translated terms in various combinations.This is a systematic review carried out upon the medical literature after a search conducted on June of 2010, including papers written in Portuguese, English and Spanish. The search for relevant references was done on databases SciELO, Cochrane, MEDLINE, and LILACS–BIREME. The publications selected for review were rated as A or B on evidence strength on the day of the review. Their authors analyzed and compared hearing aids and cochlear implants in populations of post-lingually deaf patients. The following search terms (keywords and delimiters) were used: Some of the included studies might be biased, as they were written by cochlear implant manufacturersEleven out of the 2,169 papers searched were found to be pertinent to the topic and were rated B for evidence strength  to groups of conventional hearing aid users and users of single-channel and multichannel cochlear implants. Based on outcome comparison, the authors intended to predict which patients should not be deemed good candidates for cochlear implants. The MAC test, albeit not a standardized procedure, was considered as a useful tool in advising and selecting patients for cochlear implant use.Spillmann & DillierSnik et al.vs. 53% respectively). In noisy environments, post-lingually deaf patients with 25 years or less of hearing loss also performed better.In 2001, a study by MED-EL Ltda.Bamford-Kowal-Bench (BKB) Sentence Test in noisy situations to measure speech perception and showed a significant benefit for cochlear implant users .Ching et al.Performance Inventory for Profound and Severe Loss. The CI group scored better (p < 0.01) on Visual Cues (USV), Intensity (INT), Response to Auditory Failure (RAF). No significant difference was found on Environmental Sounds (ES), Visual Cues (USNV) and Personal (PER). Note that CI patients had mean preoperative hearing in their best ears of 113 dB. On the other hand, conventional hearing aid users had mean hearing of 82 dB. Nevertheless, at the end of the study the cochlear implant group had better social hearing capabilities.Mo et al.Patient Quality of Life Form (PQLF), Index Relative Questionnaire Form (IRQF), Short Form 36 (SF-36), Hopkins Symptom Check List (HSCL-25) and Performance Inventory for Profound and Severe Loss (PIPSL). The most significant differences were observed on quality-of-life and degree of depression and anxiety (HSCL-25) between the groups of cochlear implant patients and subgroup non-CI-A. Cochlear implant patients had significantly less depression and anxiety than their non-implanted counterparts. The only significant difference between cochlear implant and conventional hearing aid users was elicited on questionnaire SF-36, in which the CI group scored better.Mo et al.Hamzavi et al.United Kingdom Cochlear Implant Study GroupMark III Health Utilities Index. The study showed acceptable cost-effectiveness for cochlear implantation in all assessed patients. It further indicated median cost-effectiveness for cochlear implants in patients affected by hearing loss for over 30 years and who were then using conventional hearing aids. Additionally, age was a factor that worsened CI cost-effectiveness, once it is estimated that older individuals have fewer years of life ahead of them.In 2004, the Bamford-Kowal-Bench (BKB) Sentence Test and the City University New York (CUNY) Sentence Test before implantation and nine months since in patients with profound hearing loss. Results have shown improved scores on both measurements nine months after surgery .On that same UKCISGp<0.05). The author observed fewer cases of depression among older cochlear implant users and of loneliness in both older and younger subjects. Older cochlear implant users were not more depressed or lonely than their counterparts with mild to moderate hearing loss who used conventional hearing aids.According to Poissant et al.Assuming that conventional hearing aid users would have better musical perception skills than cochlear implant individuals, Looi et al.Hearing loss is a problem of significant prevalence in the population that affects one's personality and socialization, possibly leading to isolation and reclusion. Hearing test results of cochlear implant patients vary. Some can communicate without the aid of orofacial reading and even talk on the phone, while others can only hear sounds from the environment, alarm buzzers or beepers, and improve their lip reading skills.Several changes are taking place as to how cochlear implant candidates are chosen, often driven the increasingly global and dynamic pace of knowledge acquisition. Patients with low frequency residual hearing in the implanted ear can undergo less traumatic procedures, avoid endocochlear trauma, and use shorter or perimodiolar electrode bundles, thus allowing them to preserve their hearing at lower frequencies and even use conventional hearing aids and cochlear implants in a combined fashion on one ear.Uma questão importante é a comprovação científica de que o IC pode trazer benefícios superiores aos das próteses auditivas convencionais, que justifiquem o procedimento cirúrgico, em termos de riscos operatórios e encargos psicossociais e financeiros ao paciente, sua família e ao sistema de saúde. Thus, cochlear implant candidates must be chosen as a function of observed outcomes and their predictive factors.According to the Brazilian Association of Otorhinolaryngology, adolescents aged 12 and over and adults with post-lingual deafness are candidates for unilateral or bilateral cochlear implants if they meet the following criteria: severe or profound bilateral sensorineural hearing loss; open form sentence recognition of 50% or less; hearing aids on both ears and patient adequately motivated to use cochlear implants and undergo speech and hearing rehabilitation.After a broad systematic review, we have been able to find methodologically distinct studies, mainly when outcomes are analyzed. However, they make it clear that cochlear implants are superior when compared to conventional hearing aids in several aspects. Some studies, such as the UKCISGIt has been found that cochlear implants allow for improvements in speech understanding and on quality-of-life, aside from significantly easing one's insertion into the social and professional worlds and reducing the onset of depression and feelings of lonelinessThe analysis of the studies included in this systematic review has enabled us to conclude that cochlear implants allow for better performance in post-lingually deaf patients when compared to conventional hearing aids as verified by audiological tests, improved quality-of-life, and overall procedure cost-effectiveness.


Unintended pregnancies pose a severe threat to the well-being of HIV-positive women and their unborn children. Factors contributing to the high incidence of unintended pregnancies include contraceptive failure, low uptake of contraceptives, and misuse of contraceptives. Despite various contraceptive options, an increased incidence of unintended pregnancies is rampant among HIV-positive women in the region of sub-Saharan Africa. This study seeks to present evidence of unintended pregnancies among women living with HIV in sub-Saharan Africa, including those using contraceptives.This study entails a scoping review to survey and interrogate the literature to provide evidence for the incidence of unintended pregnancies among HIV-positive women in sub-Saharan Africa. A proposed framework by Arksey and O’Malley will guide this scoping review. Peer-reviewed articles which address the research questions will constitute the main search. Electronic databases such as EBSCOhost, Cochrane Library, World of Science, World Health Organization (WHO) library databases, Science Direct, Google Scholar PubMed, and gray literature search will be involved. Reference list from studies included will also be searched. The investigation of articles will be done employing keywords from the studies included. The inclusion and exclusion criteria will guide two separate reviewers with the screening of abstracts and full papers. To summarize the findings from this review, thematic content analysis will be done using NVivo version 11.We expect that this review will add to the current body of knowledge on the incidence of unintended pregnancies among HIV-positive women, identify gaps for further future research, and show evidence that may contribute to strengthening the health system’s regulations, guidelines, and policies that may help prevent unintended pregnancies among HIV-positive women.10.17605/OSF.IO/EY3R5 Unintended pregnancy is a major global public health challenge that negatively impacts women, their children, and society . UnintenOver 70% of the approximately 38 million people living with HIV worldwide are in SSA, and most HIV infections occur in this region , 6. WomeUnintended pregnancy is a common phenomenon among HIV-positive women, an issue of concern . In 2018HIV-positive women are prone to a greater risk of morbidity and mortality during pregnancy and motherhood . They arAlthough the initiation of antiretroviral therapy (ART) has helped improve the quality of life of HIV-positive women , 27, it Furthermore, several studies have reported drug interactions between certain ARVs and hormonal contraceptives (HCs) –31.This scoping review mainly aims to map the evidence of unintended pregnancies occurring among HIV-positive women in SSA. We anticipate this scoping review will present the current prevalence, incidence, and risk factors of unintended pregnancies among HIV-positive women. It is also expected that this scoping review will expose literature gaps for valuable research that may influence policy decisions and means to reduce the impact of unintended pregnancies among HIV-positive women in SSA.A scoping review maps the literature on the available topic to recognize critical concepts, theories, sources of evidence, and gaps in literature , 33. It The methodological framework proposed by Arksey and O’Malley (2005) will be adopted for this proposed review. Therefore, the following five stages will be followed in this scoping review: (i) recognizing the research question, (ii) recognizing relevant studies, (iii) selection of eligible studies, (iv) charting the data, and (v) collating, summarizing, and reporting the results. This current scoping review intends to chart all research activities in this area; therefore, a quality appraisal will not be carried out.The main research question this review seeks to provide an answer to is “what is the evidence that HIV-positive women experience unintended pregnancies, including those that may be on contraceptives?”What is the evidence of the incidence of unintended pregnancies among HIV-positive women of reproductive age?What is the evidence of the incidence of unintended pregnancies among HIV-positive women of reproductive age on contraception?What are the contributing factors to unintended pregnancies among HIV-positive women of reproductive age?The research sub-questions are as follows:Published and unpublished (gray) literature will be explored on unintended pregnancies among HIV-positive women using electronic databases including Cochrane Library, World of Science, PubMed, and WorldCat. Studies reported from the year 2000 until December 2022 will be included. The Medical Subject Headings (MeSH) terms or keywords will include “HIV positive women,” “unintended pregnancies,” “contraception,” “sub-Saharan Africa (SSA),” “low- and middle-income countries,” “unplanned birth,” “unwanted pregnancy,” “unplanned pregnancy,” “unintended births,” and “unwanted births.” The appropriateness of keywords and databases will be ascertained by piloting the search strategy. To ensure no vital information is left out, keywords may be refined to include different groups of HIV-positive women, such as those using antiretrovirals (ARVs). A hand search will also be conducted of the references cited in the included studies and search on websites including World Health Organization (WHO) to identify other potentially relevant literature. Potentially relevant gray literature will be identified through specific searches of dissertations/theses  and conference abstracts. The PEO framework Table  will guiHIV-positive women of reproductive age who experienced unintended pregnanciesPublished from the year 2000 to December 2022Qualitative and quantitative studiesSub-Saharan African countries (SSA)Articles written in English languageStudies must meet the following criteria to be included:Studies without HIV-positive women of reproductive ageStudies where the researcher could not get the full-text articleStudies must have the following characteristics to be excluded:All eligible articles will be imported into Mendeley Desktop software following a comprehensive search using the keywords mentioned earlier. Duplicate reports will be identified and removed. Two separate reviewers will screen the title and abstract of the eligible articles to ascertain if they fit the review. The full-text screening will also be done see Fig. . There wRelevant information will be extracted from each of the studies included utilizing a data charting form, generated electronically using Google forms. The data extracted will consist of the following Table :Table 4DA narrative approach will present the findings from the studies that meet the inclusion criteria via thematic content analysis. NVivo version 11.0 software will be employed for the extraction themes that are relevant to the study. Collation, summary, and reporting of themes will focus on the outcomes of prevalence, incidence, and risk factors of unintended pregnancy among HIV-positive women. Also, emerging themes will be reported. These results will be described in accordance with the overall purpose of the research and help identify knowledge gaps.This proposed review is intended to map evidence for the occurrence of unintended pregnancies among HIV-positive women of reproductive age in SSA. It will also help identify risk factors for unintended pregnancies among HIV-positive women and present the burden of unintended pregnancies among HIV-positive women.Most HIV-positive women have indicated that their pregnancies were unintended , 18. SomThis review will show the evidence of unintended pregnancies among HIV-positive women in SSA. The findings of this scoping review will provide helpful information to undertake further research that will help generate the evidence that may be needed to make informed decisions regarding contraceptive use among HIV-positive women in SSA.


Primary auditory cortex is a critical stage in the human auditory pathway, a gateway between subcortical and higher-level cortical areas. Receiving the output of all subcortical processing, it sends its output on to higher-level cortex. Non-invasive physiological recordings of primary auditory cortex using electroencephalography (EEG) and magnetoencephalography (MEG), however, may not have sufficient specificity to separate responses generated in primary auditory cortex from those generated in underlying subcortical areas or neighboring cortical areas. This limitation is important for investigations of effects of top-down processing  on primary auditory cortex: higher-level areas are known to be strongly influenced by top-down processes, but subcortical areas are often assumed to perform strictly bottom-up processing. Fortunately, recent advances have made it easier to isolate the neural activity of primary auditory cortex from other areas. In this perspective, we focus on time-locked responses to stimulus features in the high gamma band (70–150 Hz) and with early cortical latency (∼40 ms), intermediate between subcortical and higher-level areas. We review recent findings from physiological studies employing either repeated simple sounds or continuous speech, obtaining either a frequency following response (FFR) or temporal response function (TRF). The potential roles of top-down processing are underscored, and comparisons with invasive intracranial EEG (iEEG) and animal model recordings are made. We argue that MEG studies employing continuous speech stimuli may offer particular benefits, in that only a few minutes of speech generates robust high gamma responses from bilateral primary auditory cortex, and without measurable interference from subcortical or higher-level areas. While the neurophysiology of primary auditory cortex has been studied for decades in animal models, there are still many unanswered questions. One of the hallmarks of primary auditory cortex in animal models is its sluggishness compared to subcortical areas, since its typical neurons time-lockns of Hz , though ns of Hz  and ongons of Hz .human primary auditory cortex, where neurophysiological recording techniques for healthy subjects are restricted to non-invasive methods, primarily electroencephalography (EEG) and magnetoencephalography (MEG). Neither EEG nor MEG has very fine spatial resolution  and so may not be able to distinguish different neural sources based purely on their anatomical origin. Both, however, have sufficient temporal resolution to distinguish typical response latencies of primary auditory cortex (∼40 ms) from subcortical (shorter latency) and non-primary (longer latency) auditory areas.Less is known about temporal processing in Beyond these commonalities, EEG and MEG have distinctive strengths and weaknesses. EEG is sensitive to neural sources throughout the brain at both low frequencies (tens of Hz) and high frequencies (hundreds of Hz) . It is tAnother consideration is that EEG’s sensitivity to most auditory sources holds for both low and high frequencies, but because of MEG’s cortical bias and because cortical responses are usually sluggish, MEG typically only captures cortical sources at low frequencies. An important counterexample, however, is the case of fast (∼100 Hz) auditory time-locked cortical responses . At thesOne newly established method to analyze neural responses to continuous speech  is tempoFast (∼100 Hz) cortical time-locked auditory responses are typically investigated using one of two different stimulus paradigms. The more time-honored paradigm is the frequency following response (FFR) , for whiThe well-established FFR paradigm  has been used to great effect with EEG to investigate midbrain responses to acoustic stimuli. Near 100 Hz, midbrain sources dominate the EEG FFR over cortical sources, and well above 100 Hz there is little to no cortical EEG FFR contribution at all . Until tOne of the limitations of the FFR paradigm is that accessing the different latencies of distinct sources may not be straightforward, since the FFR is ultimately just the evoked response to a sustained stimulus: a linear sum of overlapping responses from multiple sources with different latencies . A more High frequency (70–200 Hz) MEG TRFs were first investigated by High frequency (70–200 Hz) EEG TRFs with cortical contributions have also been recently investigated by It should not be surprising that invasive iEEG recordings had already demonstrated similar high gamma time-locked cortical responses almost a decade earlier , using cAs indicated above, a physiological window into human primary auditory cortex allows the investigation of the extent to which primary auditory cortex is influenced by higher order cortical areas. How, and under which circumstances, are primary auditory cortical responses modulated by top-down neural activity, or affected by language-specific non-auditory features of the stimulus? A related question is to what extent subcortical auditory areas might be influenced by cortical processing. Neither can be answered without first identifying the specific sources of neural activity  being modulated by distant cortical activity.Using MEG, More recently, using EEG with a continuous speech stimulus, There is additional evidence that human primary auditory cortical responses exhibit modulation arising from other cortical areas, but the effects are subtle. Using iEEG and employing selective attention to one of two competing talkers, In animal studies, top-down (task-dependent) modulation of neural activity in primary auditory cortex has been seen as far back as two decades ago . Despitepopulation model for fast acoustic features . Indeed, What is the physiological origin of the high gamma time-locked responses from primary auditory cortex? Two theories have been put forward. The first concerns the physics underlying the generators of EEG and MEG signals, which are dominantly driven by dendritic currents produced by synaptic inputs , i.e., tIn conclusion, recent advances in auditory neuroscience have opened up new non-invasive windows into the neurophysiology of primary auditory cortex. Using EEG FFR techniques, responses are dominantly subcortical but also contain strong contributions from primary auditory cortex at frequencies near 100 Hz. Using MEG FFR techniques, responses are dominantly from primary auditory cortex for frequencies near 100 Hz . EEG TRF studies have the potential to show both auditory subcortical and primary auditory cortical contributions to the time-locked high gamma responses to continuous speech, but, unlike FFR, segregated in time/latency. Finally, MEG time-locked high gamma TRF studies may hold great promise in isolating primary auditory cortical responses from other areas, due to its insensitivity to subcortical sources and its ability to differentiate competing cortical sources in both time and anatomical location.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.JZS wrote the initial draft of the manuscript. All authors contributed to the interpretations of results and discussions, were involved in manuscript revision, and approved the final version.


Introduction: Ventriculoperitoneal (VP) shunt placement is one of the most common treatments for pediatric hydrocephalus. However, device failures often occur, requiring operative revision of either the intraventricular or intraperitoneal shunt catheters. Historically, shunt placement was performed via laparotomy, but there has been a trend towards laparoscopic-assisted placement of the intraperitoneal portion of the shunt. We examined the outcomes of laparoscopic-assisted versus open VP shunt placement utilizing a local institutional retrospective review.Methods: Single institution 2012-2017 retrospective review of all cases was performed. Patients were divided into two groups - laparoscopic and open. Thirty-day outcomes, patient age, surgery performed, surgical control time (SCT), length of stay (LOS), and readmission were analyzed. Results: Cohort analysis inclusion criteria included 188 patients. The cohort analysis showed both decreased laparoscopic-assisted SCT  and postop complications . There was no significant difference in surgical site infection or readmission rates. Conclusion: Local analysis show advantages for laparoscopic-assisted VP shunt placement over open single surgeon techniques with decreased SCT, LOS, and unplanned interventions. Hydrocephalus is a significant problem in the pediatric population, occurring at a rate of 0.2-1.2 per 1000, and its management leads to 40,000 admissions a year and $2 billion in cost . Recent Given this high rate of shunt complications, innovations in distal shunt placement have been sought to minimize morbidity following VP shunt placement. Laparoscopy for placement of the peritoneal catheter was first described in the early 1990s . Known bRecently, studies have demonstrated the feasibility of laparoscopically assisted placement of the distal portion of the VP shunt ,10. HoweA retrospective cohort of new VP shunt placements performed at a single surgical center over a time period from 2012 to 2017 underwent chart review. Patients were included if they were under the age of 22 years and were a first-time VP shunt placement for any cause of hydrocephalus. Patients were excluded if they had incomplete data, were older than the age of 22 years, or were subdural-peritoneal shunt insertions. Although our primary focus is on postoperative patient complications, because operative time was a variable of interest, we aimed to standardize the procedural intervention as much as possible.Patients were classified based on the surgical approach to placement of the peritoneal portion of the catheter. The open surgical cohort received a VP shunt from a neurosurgeon performing both the ventricular access and obtaining peritoneal access for the catheter using an open or mini-laparotomy in the traditional fashion. The laparoscopic cohort involved the addition of a pediatric surgeon to obtain peritoneal access and placement of the shunt catheter utilizing laparoscopic methods. Retrospectively collected perioperative data was obtained from the electronic medical record. Demographic data included sex, age, and insurance. Perioperative data included operative time, total and postoperative length of stay (LOS). Outcome measures primarily assessed complication rates, need for readmission or reoperation within 30 days, and the 90-day infection rate. Informed consent was waived after evaluation by our local investigational review board (IRB) and IRB approval is under file #11-18-28E.Statistical analysis was performed using STATA statistics and data analysis . Statistical significance was set at p < 0.05. Confidence intervals of 95% were also used in analysis where appropriate.Population characteristicsA total of 230 consecutive VP shunt insertions were available for review. Thirty-seven patients were excluded for incomplete data, and an additional five patients received subdural-peritoneal shunt insertion and were excluded to maintain similar technical procedure. Therefore, 188 patients met all inclusion criteria and were analyzed. A total of 90 (48%) patients underwent laparotomy with a neurosurgeon alone, and 98 (52%) patients underwent laparoscopy in conjunction with a pediatric surgeon. Patient characteristics between both groups were statistically similar (Table Clinical outcomesClinical outcomes of our cohort analysis are included in Table The overall benefit of laparoscopic-assisted ventriculoperitoneal shunt placement as an initial surgical approach has been a contested point of discussion for several decades. Studies have previously shown the benefits of a laparoscopic-assisted approach including decreased surgical time, reduced risk of ileus, ease of access in obese patients, and decreased risk of adhesions ,4,11,12.The cohort portion of our study is additive to the currently available literature in that we present 188 first-time ventriculoperitoneal shunt placements, performed consecutively, with direct analysis of outcomes between single-surgeon laparotomy and co-surgeon laparoscopy assisted placement. In a search of the literature, few studies have directly compared outcomes between mini-laparotomy and laparoscopic-assisted procedures -21. AddiOur study was not designed to address the impact of laparoscopic-assisted VP shunt placement on overall costs. However, we know that many of the outcomes in our study do influence the cost of cases. While Catapano et al. were able to demonstrate a financial benefit in total hospital costs in a subgroup analysis of NPH patients , GravbroOverall, when analyzing our institutional outcomes, we see a substantial improvement in the postoperative outcomes of patients undergoing laparoscopic assisted VP shunt placement. This has become the standard practice in our institution as we believe it provides the best care for patients in our setting. The limitations of our study include the small sample size in our institutional cohort and variation in length of stay due to patients ranging from neonatal infants with significant medical comorbidities to patients with primary hydrocephalus without medical comorbidities that could impact length of stay. However, we believe this analysis provides a valuable addition to the literature on the benefits of laparoscopic-assisted VP shunt placement.Shunt placement is one of the most common procedures in pediatric neurosurgery. Small technical changes can significantly impact the overall outcome and some of the simplest such as optimal skin sterile prep or hand washing procedures have been evaluated in the past. We found that patients undergoing laparoscopic assisted VP shunt placement have substantially improved surgical outcomes. This includes a trend toward reduced surgeon control time, a shorter post-operative length of stay, and less than half the rate of unplanned interventions. Collaboration between pediatric neurosurgeons and pediatric surgeons facilitates care and improves overall patient outcomes.


Sociodemographic predictors of employee alcohol use are well established in the literature, but knowledge about associations between workplace factors and alcohol use is less explored. The aim of this study was to explore whether workplace factors were associated with employee alcohol use .Cross-sectional study. Linear and binary logistic regression analyses.Heterogeneous sample of employees (workers and supervisors) from 22 companies across geographical locations and work divisions in Norway.Employees (N=5388) responded on survey items measuring workplace factors and alcohol use.Data on alcohol use were collected with the Alcohol Use Disorders Identification Test (AUDIT). Consumption was measured with the AUDIT-C (the first three items), and alcohol-related problems were operationalised as a sum score of 8 or higher on the full 10-item AUDIT.Higher levels of alcohol consumption were associated with more liberal workplace drinking social norms , working full-time , working from holiday home , being a supervisor , having supervisors with less desired leadership qualities , shorter working hours , higher workplace social support  and higher income . Alcohol-related problems were associated with more liberal workplace drinking social norms  and shorter working hours .Workplace drinking social norms were the supremely most dominant predictor of both consumption and alcohol-related problems. Results suggest that some workplace factors may play a role in explaining employee alcohol consumption, although the predictive ability of these factors was limited. This study points to the importance of drinking social norms, workplace drinking culture and leadership for understanding employee alcohol use. This study explores the importance of a wide range of workplace factors for employee alcohol use in a large and heterogeneous sample by means of a validated alcohol screening instrument.This study uses individuals’ overall degree of consumption, as well as odds of having a drinking pattern that may induce alcohol-related problems, as outcomes.Study limitations include a low response rate, the use of self-reported data and a cross-sectional design that precludes causal inferences.Alcohol consumption is associated with detrimental health outcomes.5 8Alcohol is the most commonly used and misused psychoactive substance in the workforce.19–21According to Frone’s model of employee substance use,Studies have demonstrated that workplace alcohol use climate may affect the drinking level of employees,et al revealed that working part-time (as opposed to full-time) was associated with higher alcohol use per week, but unrelated to prevalence of binge drinking episodes and alcohol-related consequences.Systematic reviews generally point to longer working hours being associated with higher alcohol consumption.Travels and worksite factors may refer to the degree to which employees are subjects of direct monitoring at the workplace. According to Roman and Trice,47Research on the importance of job demands and support factors  for employee alcohol use has yielded variable results. It has been advocated that visible and active supervisors constitute a protective factor for alcohol use among workers,56In summary, evidence on associations between workplace factors and employee alcohol use stands out as complex and nuanced. Moreover, few studies have explored the importance of workplace factors for alcohol consumption as well as for odds of alcohol-related problems in heterogeneous employee samples by means of validated alcohol screening instruments. Individuals’ overall degree of consumption, and their odds of having a drinking pattern that may induce alcohol-related problems, constitute two different—yet equally important—ways of conceptualising alcohol use that can be differentially associated with workplace factors. Knowledge about associations between workplace factors and employee alcohol use is important as an aid in determining the extent to which employers should make workplace-based alcohol health promotion and prevention interventions an overall priority, and for determining which elements should be emphasised in such interventions. Hence, further research is warranted.The aim of this study was to explore whether workplace factors  were associated with employee alcohol consumption and odds of having alcohol-related problems in a heterogeneous sample of employees in Norway.This cross-sectional study explored associations between workplace factors and alcohol use in a heterogeneous sample of employees (workers and supervisors) in 22 companies in Norway , across geographical locations. The study was part of the Norwegian national WIRUS Project .The researchers recruited companies through three occupational health service units’ client lists in collaboration with the addiction competence environment KoRus Stavanger. Individual-level inclusion criteria were: (1) age 16–72 years; (2) status as employee, regardless of sector, work division or geographical location; and (3) basic understanding of the Norwegian language. Our definition of employees (criteria 2) included white-collar professional roles, business owners), blue-collar (labourers and skilled trade roles) and pink-collar employed .Data from participants were collected between October 2015 and October 2019. Hence, each participant was measured once (between 2015 and 2019), and all data were collected prior to the COVID-19 pandemic. We predefined that a statistically satisfactory sample size had to conform with the formula N>50+(8×number of predictors/covariates),62alcohol consumption, was measured with the first three AUDIT items , often referred to as the AUDIT-C.alcohol-related problems, was measured as a dichotomous categorical variable , based on a cut-off of 8 on the full 10-item AUDIT .7The outcomes were measured with the Alcohol Use Disorders Identification Test (AUDIT). The AUDIT is an alcohol screening instrument, developed by the WHO,working hours , work schedule , job position , job size  and income .Employment characteristics included psychological job demands, job control, workplace social support and supervisors’ leadership qualities. Psychological job demands (five items), job control  and workplace social support  were measured with the Job Content Questionnaire (JCQ),Job demands and support factors included employees’ perceptions of job travels , working from home  and working from holiday home .Travels and worksite factors (reflecting degree to which employees are under direct monitoring by coworkers and supervisors) were measured in terms of whether the job included drinking social norms, measured with the four items from the Drinking Norms Scale (DNS) that directly relates to the work situation.Workplace alcohol use climate was conceptualised as employees’ perceptions of An overview of workplace factors is presented in gender , age (years), educational attainment , living status , marital status  and number of children. Due to the cross-sectional data in this study being collected over a period of approximately 5 years, year of data collection  was included as an additional covariate.The following sociodemographic factors, which have demonstrated significant associations with alcohol use in a Norwegian employee population,Descriptive analyses were performed on the main study variables (outcomes and predictors) and presented in terms of means, standard deviations, frequencies and percentages, as appropriate.Main analyses were conducted in two stages: model selection and predictor comparison.2 values for all possible subset models in a multiple regression, with additional contribution of each predictor. A predictor is considered to be more important than its competitors if its predictive ability exceeds the others in all subset regressionsIn the predictor comparison stage, the relative importance of predictors that demonstrated significant associations with the outcomes (p<0.05) in fully adjusted models in the model selection stage was estimated by means of dominance analysis (DA).Significant results were defined as p<0.05.Employee representatives and other relevant stakeholders were included in the WIRUS Project’s reference group. Design, research questions, recruitment procedures and choice of outcome measures in the project have been informed by priorities, experience and preferences expressed by the reference group.Job control=3.0; MSocial support=3.2; MPsychological job demands=2.2). Employees’ mean rating of supervisors’ desired leadership qualities was 2.9 . On average, employees reported somewhat more restrictive than liberal workplace drinking social norms (M=2.2 on the DNS ranging from 1 to 4). Descriptive statistics for the main study variables are presented in Approximately 1 out of 10 employees (11.5%) reported alcohol-related problems (AUDIT ≥8). The sample’s mean score on alcohol consumption was 3.2 . The majority of employees were workers  with full-time  day job schedules without weekends . Employees’ average gross annual household income was 1 012 000 Norwegian Kroner (approximately £83 587), and a minority of employees had a job that included job travels , working from home  and working from holiday home . Job control and workplace social support were rated higher than psychological job demands  are presented in the 10.1136/bmjopen-2022-064352.supp1Supplementary dataAssociations between workplace factors and alcohol consumption are presented in As shown in Higher consumption was associated with shorter working hours, being a supervisor (vs a worker), working full-time (as opposed to part-time), higher income, higher levels of workplace social support, having a supervisor with less desired leadership qualities, working from holiday home and more liberal workplace drinking social norms. The remaining workplace factors either failed to demonstrate significant relationships with alcohol consumption altogether , or failed to reach statistical significance in the fully adjusted model .The relative importance of the eight significant workplace factors for explaining the variance in alcohol consumption is presented in Based on average contribution to the explained variance in alcohol consumption, drinking social norms (17.92%) was the dominant predictor, followed by job size (0.45%), working from holiday home (0.16%), job position (0.13%), leadership qualities (0.07%), working hours (0.05%), workplace social support (0.02%) and income (0.00%). In the fully adjusted regression model , model 5Associations between workplace factors and odds of having alcohol-related problems are presented in As shown in Elevated odds of alcohol-related problems was associated with shorter working hours and more liberal workplace drinking social norms. The remaining workplace factors either failed to reach statistical significance in the fully adjusted model , or were unrelated with alcohol-related problems altogether .Dominance analysis  revealedA 1-hour increase in working hours was associated with reduced odds of alcohol-related problems by a factor of 0.94. A one-unit increase on the drinking social norms scale  was associated with increased odds of alcohol-related problems by a factor of 3.52.Main results are summarised in Eight out of 16 workplace factors were not significantly related to either alcohol consumption or alcohol-related problems. Six workplace factors were significantly associated with alcohol consumption but not with alcohol-related problems . Two workplace factors were significantly related to both alcohol consumption and alcohol-related problems . With the exception of drinking social norms , associations between workplace factors and the alcohol outcomes were quite weak see  and mostThe aim of this study was to explore whether workplace factors were associated with alcohol use in a heterogeneous sample of employees in Norway.Drinking social norms emerged as the supremely most important predictor of both alcohol consumption and alcohol-related problems. More liberal attitudes toward the role of alcohol in workplace settings were associated with having a higher consumption as well as elevated odds of having alcohol-related problems. Drinking norms prescribe the level of consumption that is considered appropriate or tolerable in specific social contexts, and employees’ drinking norms may reflect the workplace drinking culture.78Interestingly, longer work hours were associated with lower consumption but also with higher odds of having alcohol-related problems. These findings may, intuitively, stand out as somewhat contradictory, insofar that one may expect alcohol consumption to be closely associated with alcohol-related problems. We did find a considerable and statistically significant correlation between the two alcohol outcomes. However, the strength of the correlation was in the upper layer of moderate strength see , which i29–34Higher income was associated with higher alcohol consumption. This finding is consistent with earlier studies emphasising that higher socioeconomic status is related to higher levels of consumption.Working full-time (as opposed to part-time) was associated with elevated alcohol consumption among employees. This finding contradicts earlier research from the USA that has found that part-time workers had higher weekly consumption than full-time workers.Workers consumed less alcohol than supervisors, which is in line with some earlier studies.23 31 34 35Worksite factors  and job travels have in some studies been linked with elevated alcohol consumption.83 84Having supervisors with desired leadership qualities was associated with lower alcohol consumption. Supervisors who are highly rated on desired leadership qualities  affecting both job size and drinking pattern. However, the aim of this study was not to unravel causal relationships but was explorative in nature. The cross-sectional design may also have played a role in explaining why observed effect sizes were quite small. Potential effects of some workplace factors on broad indicators of alcohol use and problems may be temporary and/or short lived, rendering it difficult to fully capture true effects without more robust research designs that involve longitudinal measures.The sample was large and consisted of a heterogeneous selection of employees across work sectors, divisions, levels and geographical locations. The sample size well exceeded the a priori defined criteria,This study was solely based on self-reported data from employees, which may involve risks of measuring bias . In particular, one may assume that alcohol use may have been underestimated, insofar that studies have demonstrated a discrepancy between self-reported consumption and actual alcohol sales.Among the workplace factors explored in this study, drinking social norms emerged as the supremely most important predictor of employee alcohol use. Hence, results from our study call attention to the importance of workplace drinking cultures to understand employees’ level of alcohol consumption and odds of experiencing alcohol-related problems. Furthermore, other significant predictors of employee alcohol use support the notion of emphasising workplace drinking culture. Perceiving higher levels of social support and being full-time employed were both associated with higher consumption levels and can both be conceptualised as proxies of social integration at the workplace, thereby indicating the importance of workplace culture. Moreover, results indicate the importance of supervisor behaviour. Supervisors consumed more alcohol than workers, and having supervisors with high levels of desired leadership qualities was associated with lower levels of consumption among employees. Efforts aimed at reducing alcohol consumption and alcohol-related problems among employees may include fostering less liberal drinking cultures at workplaces in combination with desired leadership qualities among supervisors. Raising supervisors' awareness of their influence on workplace drinking culture may be serviceable. Prosocial supervisors may stimulate workplace cultures that discourage or inhibit drinking in work-related situations and may also foster social relationships and trust that enable establishment of routines for early identification and aid for employees who may benefit from alcohol prevention interventions.In a systematic review,Evidence on associations between workplace factors and employee alcohol use still stands out as complex and nuanced, and this study showed that only marginal proportions of the variance in alcohol consumption and alcohol-related problems were explained by employment characteristics, job demands and support factors and travels and worksite factors. Hence, further research is warranted. Results from this study suggest the application and further development of interventions, for example, Workplace Health Promotion Programs, targeting workplace drinking culture and leadership. Effects and implementation of such interventions should be explored by means of robust research designs, such as prospective cohort studies and cluster randomised controlled trials. Future research may also benefit from explorations of potential interactions between individual and workplace factors, including mediators and moderators, which were beyond the scope of our study.Knowledge about workplace factors associated with employee alcohol use is important as an aid in determining the extent to which employers should make workplace-based alcohol prevention interventions an overall priority. This study, conducted in a large and heterogeneous sample of employees in Norway, points to the importance of drinking social norms, workplace drinking culture and leadership.


Atomic force microscopy (AFM) and high-speed scanning have significantly advanced real time observation of biomolecular dynamics, with applications ranging from single molecules to the cellular level. To facilitate the interpretation of resolution-limited imaging, post-experimental computational analysis plays an increasingly important role to understand AFM measurements. Data-driven simulation of AFM, computationally emulating experimental scanning, and automatized fitting has recently elevated the understanding of measured AFM topographies by inferring the underlying full 3D atomistic structures. Providing an interactive user-friendly interface for simulation AFM, the BioAFMviewer software has become an established tool within the Bio-AFM community, with a plethora of applications demonstrating how the obtained full atomistic information advances molecular understanding beyond topographic imaging. This graphical review illustrates the BioAFMviewer capacities and further emphasizes the importance of simulation AFM to complement experimental observations. DirectlHowever, magic experiments do not exist, and AFM observations also have several limitations. Since the scanning tip probes only morphological changes within the accessible molecular surface, important motions may be missed and thus information about functionally relevant conformational couplings. Due to the tip-size being much larger than the atoms it interacts with, measured topographies do not resolve structural details on the sub-nanometer range. Furthermore, limitations in the scanning speed prevent to monitor a molecular movie of continuous conformational changes while HS-AFM records rather stroboscopic snapshots of biomolecular function.While technological developments towards building next-generation HS-AFM with improved scanning speed are currently undertaken, intrinsic limitations inevitably remain. As a result of this, post-experimental computational analysis of AFM images is becoming increasingly important to facilitate the interpretation and understanding of measurements. For quantitative analysis of AFM topographies convenient software plugins within the ImageJ  platformTopical developments are aimed to predict 3D biomolecular structures from topographic AFM imaging. While docking of static structures into experimental topographies has been addressed previously , more soDevelopment of the BioAFMviewer with its user-friendly interactive interface and rich functionality has opened the opportunity for the broad Bio-AFM community to employ data-driven simulation AFM and facilitate the interpretation of experimental observations . Indeed,For a plethora of applications, ranging from single molecular machines, protein filaments, to large-scale assemblies of protein lattices, simulation AFM and automatized rigid-body fitting has been demonstrated to advance the understanding of measured AFM topographic images by providing atomistic-level information . Simulat2https://www.bioafmviewer.com.Of major importance to us was that the computational procedure of simulated AFM scanning, the mathematical methods for automatized fitting available biomolecular structures to experimental AFM images, and the tools for quantitative topography analysis are embedded within a highly versatile user-friendly graphical interface. Equally important was to share our developments with the broad Bio-AFM community to strengthen interdisciplinary research. The BioAFMviewer provides a complete software package which is freely available at 2.1The idea of simulation AFM is to computationally emulate experimental scanning of biomolecules to translate their available high-resolution molecular structures into simulation AFM topographic images which can be correlated with experimentally obtained AFM images. Central to the BioAFMviewer software is an integrated molecular viewer visualizing the loaded PDB structure in any 3D orientation and synchronized computation and visualization of the corresponding simulation AFM image .Fig. 1Si2.2Integrated into the BioAFMviewer is a module which employs simulation AFM for automatized rigid-body fitting of available biomolecular structures to experimental AFM images. Thus, it becomes possible to identify the 3D atomistic structure behind measured AFM topographies . This op2.3A plethora of applications, ranging from single molecular machines, protein filaments, to large-scale assemblies of protein lattices, demonstrate how the full atomistic information obtained from simulation AFM and efficient automatized fitting advances the molecular understanding beyond original resolution-limited topographic AFM imaging .Fig. 3Ap2.4The BioAFMviewer contains a topography analysis toolbox which allows comparison of simulated and measured topographies. Several applications demonstrate that by a more detailed analysis, simulation AFM allows the validation of measurements and quantitative feature assignment within resolution-limited measured topographies .Fig. 4To2.5Applications of simulation AFM go beyond fitting of equilibrium data to infer atomistic structures from AFM imaging. Molecular movies of biomolecular conformational dynamics obtained from simulations can be processed in the BioAFMviewer to generate simulated AFM experiments . Simulat3Simulation AFM plays an increasingly important role to complement experimental AFM imaging. In fact, given the enormous amount of high-resolution protein structures already available from the Protein Data Bank, together with the rapidly increasing data from AlphaFold predictions, there is no excuse not to employ simulation AFM to facilitate the interpretation of resolution-limited AFM observations by inferring 3D atomistic information from measured topographies. The BioAFMviewer platform has become a standard tool used by the Bio-AFM community worldwide. The increasing number of applications evidence the importance of simulation AFM. Further software developments are actively pursued and closely coordinated by intense collaborations with experimental groups.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Hypermature cataract is a form of late-stage cataract progression that can lead to a variety of complications. Spontaneous capsular rupture with lens nucleus displacement in hypermature cataracts has rarely been reported. We describe 2 cases of spontaneous dislocation of the lens nucleus in a hypermature cataract and perform a review of the literature on this complication.We report 2 rural men aged 50 and 76 years with deteriorating vision.The final diagnosis was senile hypermature cataract with dislocation of the lens nucleus in both patients and secondary glaucoma for the second patient.During admission, both patients complained of deteriorating vision. Slit-lamp examination showed lens nucleus dislocation into the anterior chamber. The 50-year-old patient exhibited a residual lens capsule and a turbid cortex, with a normal anterior chamber and intraocular pressure. The 76-year-old patient presented a shrunken and ruptured capsule and no cortex in the pupillary area, mild inflammation in the anterior chamber, and high intraocular pressure. Both patients underwent intracapsular cataract extraction combined with anterior vitrectomy and achieved good postoperative recovery.Lens nucleus dislocation in hypermature cataracts can be seen in clinical practice, particularly in underdeveloped areas. Early recognition and surgery can improve vision. Herein, we describe 2 cases of spontaneous dislocation of the lens nucleus in a hypermature cataract. We review the literature on lens nucleus dislocation focusing on the diagnosis and treatment of hypermature cataracts in these specific cases.With improvements in the standards of living and health awareness, many cataract patients have been cured by surgical procedures in the early and middle stages of the disease. However, in some remote and less developed areas, hypermature cataracts continue to be a problem. Hypermature cataracts can lead to many complications, including lens-induced uveitis,This study adhered to the tenets of the Declaration of Helsinki and was approved by the Human Research Ethics Committee of the First People’s Hospital of Kashi (ethics no. 2021ksyd-18). Both participants provided written informed consent. The clinical timeline of the cases is shown in Figure A 50-year-old man from a rural area was admitted to the Department of Ophthalmology of the First People’s Hospital of Kashi due to poor left eye (LE) vision that had been deteriorating over the last 12 years, with recent worsening of symptoms during the previous month. No eye pain or redness was noted upon admission. The patient reported “gastric repair” for “gastric perforation” 3 years prior but denied having a history of eye trauma or other diseases. Otherwise, personal and family histories contained no relevant details.2 with 38% hexagonal cells. Optical coherence tomography (OCT)  and B-scan ultrasonography  revealed a flat retina in the LE. All laboratory examinations were within normal ranges.On examination, the best corrected visual acuity was 20/20 in the right eye (RE), and counting fingers at 5 cm in the LE with a normal visual field and red-green color perception. The intraocular pressures (IOPs) measured with Goldmann applanation tonometry were 12 mm Hg and 8 mm Hg in the RE and LE, respectively. Slit-lamp examination showed no abnormalities of the RE , xenobiotic detoxification (NAT2), DNA repair (ERCC2), folate metabolism (MTHFR), lactose metabolism (LCT), RNA demethylation (FTO), lipid/cholesterol transport (APOE4), kinesin/microtubule motor transport (KLC1), and one of unknown function (ARCC1). The patient in case 1 had a hypermature cataract at a relatively young age, which is an atypical type of cataract. Whether it was complicated by genetic factors or systemic diseases requires a complete system evaluation and genetic examination. However, due to the patient’s financial constraints, we did not carry out genetic testing.It should be noted that case 1 was a 50-year-old man with no history of trauma, metabolic abnormalities, or systemic symptoms that could explain his eye condition. A hypermature cataract or spontaneous rupture of the lens capsule, such as that occurring with polar cataract, In these 2 cases, patients underwent intracapsular cataract extraction because of an incomplete posterior capsule. Anterior vitrectomy was performed to relieve tension on the posterior retina and prevent macular edema. Additionally, secondary intraocular lens implantation was considered to avoid severe postoperative inflammatory reactions and for accurate measurement of the intraocular lens.Cataract surgeries have evolved to include the development of intracapsular extraction, extracapsular extraction, and phacoemulsification. The surgical method should be individualized according to the local medical facilities, the skills of the surgeon, and the actual situation of the patient. Intracapsular cataract extraction has largely been replaced by modern cataract surgery due to many intraoperative and postoperative complications of the former procedure, including vitreous loss, cystoid macular edema, and corneal endothelial cell injury. However, intracapsular cataract extraction is still occasionally used in some underdeveloped areas. In some remote and underdeveloped rural areas in China, patient demand for vision correction is low. Seeking treatment for the affected eye may be delayed when vision in the contralateral eye is good. Unfortunately, hypermature cataracts may cause serious complications and lead to permanent visual impairment. These adverse results can be prevented by early cataract surgery, which suggests that future work should focus on raising awareness of eye health, improving the rate of physical eye examinations, and taking effective measures to prevent the occurrence of hypermature cataracts.In low- and middle-income countries, hypermature cataracts still account for a large proportion of cataract surgeries.Hypermature cataract remains a relatively common occurrence in underdeveloped areas, which can lead to many complications. Spontaneous capsular rupture with lens nucleus displacement in a hypermature lens can be seen in clinical practice. Intracapsular cataract extraction and anterior vitrectomy can easily correct this issue, and secondary intraocular lens implantation can improve vision.Conceptualization: Jie-Ying Guan, Ying-Ting Zhu and Ye-Hong ZhuoData curation: Yun-Cheng Ma and Aizezi WumaierInvestigation: Jie-Ying GuanWriting—original draft: Jie-Ying GuanRevise and editing: Ling-Ling Xie, Mireayi AizeziFund acquisition: Aizezi Wumaier


Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy  to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune, and vasculature components across 323 fields of view. Single-cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core fields of view of responders. Integrating single-cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the tumor-centric cellular neighborhood (TCCN) based on anatomic subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytic framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy.Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion, including the downregulation of HLA-I, potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet, the Findings from this work propose a novel approach to resolving clinical heterogeneity of the TIME by objectively quantifying the cellular interactions occurring in metastatic melanoma lymph node tissue utilizing multiplex immunofluorescence. This study provides an analytic and biologically derived unit of measure, the TCCN which is customizable for studying critical paracrine interactions within spatially preserved tissue of various cancers and across the spectrum of multiplex imaging modalities. Treating metastatic cancer with immunotherapy has dramatically altered the clinical landscape of medical oncology, especially for patients with metastatic melanoma. And yet, clinical responses to immune checkpoint inhibitors (ICI) remain suboptimal and challenging to predict . SpecifiThe influence of the tumor immune microenvironment (TIME) on cancer progression, metastasis, and therapeutic responsiveness has been studied by numerous groups and is now recognized as a pillar in the hallmarks of cancer . HistoriFor metastatic melanoma, and most solid tumor malignancies, the lymph node is the most common, first site of metastatic dissemination . ServingTo deconstruct the inherent complexity of the TIME, bioimaging modalities have rapidly evolved to enable deep profiling at single-cell resolution. These technologies were first applied to liquid biopsies and single-cell suspensions but have more recently been developed for formalin-fixed paraffin-embedded tissue . CurrentIn this study, we applied a cyclic multiplex immunofluorescence (MxIF) platform to archival lymph node tissue to characterize the TIME of metastatic melanoma in 14 treatment-naïve patients with clear objective responses to anti-PD1 therapy. Utilizing a 35-analyte panel, the MxIF platform enabled single-cell, quantitative evaluation of tumor and immune cell subsets within microanatomic regions of the metastatic lymph node including the tumor core, tumor margin, and lymphoid tissue. To interrogate paracrine interactions of tumor cells derived from these distinct regions, we developed a biologically derived spatial metric, the tumor-centric cellular neighborhood (TCCN). This spatial metric was applied to the metastatic lymph node to evaluate how differences within the tumor-immune paracrine landscape relate to distinct anatomic regions, molecular features of individual tumor cells and clinical outcomes in patients undergoing ICI therapy.Thirty treatment-naïve patients with metastatic melanoma who received anti-PD1 immunotherapy as part of their treatment course were selected for MxIF analysis in the current study. From this initial cohort, 14 cases with clear objective responses as defined by RECIST, complete response (responders) versus progression (nonresponders) to immunotherapy were selected for subsequent analysis. Analogous to the clinical response rates reported to anti-PD1 treatment in stage IV melanoma , our stuThe panel was designed with 35 antibodies capable of elucidating the phenotypic and functional status of the tumor, immune, vascular, and structural components of the lymph node TIME. A summary of all antibodies is provided in After image acquisition the antibodies were evaluated again for overall performance in the study-specific tissue including staining specificity, signal-to-noise ratio, autofluorescence (AF) removal, and artifact detection. As a result, three antibodies were removed from the final analysis. Antibody panel design enabled phenotypic and functional differentiation of tumor and immune cell subsets as detailed in our classification pipeline.3 (pH 8.5), and dye conjugate and incubated for 60 minutes in the dark at room temperature. Following the conjugation, the reaction was loaded on a Zeba desalt spin column (Thermo Fisher Scientific) to remove unbound fluorophore, and the final concentration and dye:protein ratio measured by Nanodrop. Following conjugation, the antibody solution was adjusted to a final concentration between 150 and 300 μg/mL and stabilized with 1% BSA and 0.45% sodium azide.Rabbit and mouse anti-human antibodies were first purified on HiTrap Protein A or Protein G columns respectively to remove additives and stabilizers. Buffer exchange and concentration was conducted using Amicon 30K filters and purified antibody was resuspended in PBS buffer at a final concentration of 1 mg/mL. Direct conjugation to Cy2-, Cy3-, and Cy5-Bis NHS Ester dye fluorophores  was done according to manufacturer's instructions. The conjugation reaction was prepared with a minimum loading of 35 μg of protein, 10% w/v of NaHCOSlide preparation and antigen retrieval was done as described previously . Briefly2). Following FOV selection at the 10× objective, background imaging at the 20× objective was conducted for each FOV at each fluorescence channel .Images were acquired using the INCell Analyzer 2500HS . Following initial DAPI staining, imaging at a 10× objective at all 798 positions across the glass slide area were stitched to provide a whole slide image. A single region of interest including the entire tissue specimen was then selected and a virtual H&E image was created with the DAPI and Cy3 channels. Using RhedEye software, a clinical pathologist annotated the metastatic lymph node tissue to enable selection of fields of view (FOV) that satisfy three anatomic regions of interest: tumor core, tumor margin, and lymphoid tissue . Each FO3, pH 11.2 solution for two washes of 15 minutes each. Following the inactivation, the slides were washed three times in PBS, briefly incubated with DAPI before a final PBS wash and then coverslipped as described previously. Imaging of the dye inactivation round was acquired between each stained round as described below. After image acquisition of a stained or dye inactivation round, the coverslip was removed by inverting slides in a PBS bath with gentle agitation.Staining with multiple antibodies was achieved by alternating rounds of dye inactivation imaging with stained tissue imaging. For immunofluorescence staining, slides were incubated with an antibody cocktail (200 μL) diluted in PBS + 3% BSA  for 1 hour at room temperature in a humidified chamber. Following three PBS washes, the slides were coverslipped for imaging (see Multiplex Imaging Acquisition and Preprocessing section below). Following imaging, dye inactivation was performed by incubating slides in 0.5 mol/L NaHCOImages were acquired using the INCell Analyzer 2500HS scanner  according to manufacturer's protocol . BrieflyImage files created from acquisition include a raw image, registered image, bleach image, and autofluorescence (AF) removed image. Per-pixel image subtraction of the dye inactivation image from the stained image in the corresponding round was performed for AF removal. After the digital removal of AF, image normalization was applied, which scaled the intensities according to the minimum exposure time in the dataset and then image subtraction was computed per pixel between the stain to dye inactivation captures. This step includes the algorithm accounting for dark pixel intensity offset  dimension reduction, FOVs were grouped by pathologist assigned label  algorithm . This alThe data generated in this study are available within the article and its Supplementary Data files. Supporting data generated in this study are available upon request from the corresponding author.Increasingly, dynamics of the TIME have been recognized for their contribution to variable clinical responses to immunotherapy in the metastatic setting. To quantitatively profile the complexity of the TIME in spatially resolved metastatic tissue, we adapted a cyclic MxIF imaging approach to evaluate surgical excisional lymph node biopsies obtained from treatment-naïve patients with metastatic melanoma who underwent subsequent immunotherapy . ThroughAbundance plots summarize the cell counts and proportions of each cell class at the FOV level across the distinct regions of tumor core, margin, and lymphoid tissue. Expectedly, abundance plots reflected the anatomic region sampled with tumor core FOVs displaying the highest tumor class abundancies, lymphoid tissue FOVs displaying primarily immune cell subsets, and interface FOVs comprised of a mixture of all classes . Next, sAn established mechanism of immune evasion employed by tumor cells with potential implications in poor response to ICI, is the downregulation of HLA-I expression on the tumor cell surface . While aBuilding from single-cell quantification at the resolution of anatomic subregions and functional status of tumor cells, we next considered the higher dimensional spatial arrangements of the individual cells and their immediately adjacent cell neighbors to further differentiate distinct TIME. To evaluate the paracrine network of interactions of cells within close proximity to each other, we adapted the method of CNA by modeling a micro-niche surrounding each tumor cell in a given FOV, termed a tumor-centric cellular neighborhood (TCCN). Neighborhood-based analysis was dependent on the following two criteria: (i) the center (index) cell must be classified as a tumor cell and (ii) the neighborhood must be occupied by at least one additional cell beyond the index cell . By thesUtilizing TCCN as the spatial metric of the TIME landscape, we next sought to evaluate the differences in immune composition in TCCNs derived from distinct anatomical subregions of the lymph node: the tumor core and margin. The percent of TCCNs populated by different immune cell phenotypes was determined for tumor core and tumor margin in responders and nonresponders and revealed that overall TCCNs derived from responders had an increased percentage of TCCNs populated with immune cells compared with nonresponder counterparts. Specifically, in core-derived TCCNs, responders had a significant increase in cytotoxic T cells, macrophages, and neutrophil-populated TCCNs while margin-derived TCCNs showed an increase in B cells, Th cells, regulatory T cells, and neutrophil-populated TCCNs in responders . OverallMapping this observation within the spatial landscape, TCCNs were plotted back to the anatomical FOV of origin to consider the dominant immune cell type within each TCCN of the tumor core and margin. Considering only the immune cell subsets that distinguished responders from nonresponders in the core and margin respectively, spatial FOVs were visualized for responders and nonresponders. Treating each TCCN as a point on the FOV, the spatial distribution of TCCN in the tumor core versus tumor margin can be visualized. Compared with the corresponding virtual H&E image, TCCNs were more evenly dispersed throughout tumor core FOVs while the spatial localization of TCCNs to the tumor and immune-interfacing edge is apparent in the tumor margin FOV. Notably, the responder TCCNs illustrated more frequent integration among immune cell types within the same TCCN, while the nonresponder TCCN suggests primarily macrophage occupied TCCN comprise the tumor margin at the interacting edge .pos and HLA-Ineg TCCNs while an increase of macrophages was only observed in HLA-Ineg iTC TCCNs of responders. In the tumor margin, Th cells and neutrophils more frequently populated responder TCCNs irrespective of HLA-I iTC status, while B cells, macrophages, and regulatory T cells were also increased in HLA-Ineg iTC TCCNs of responders. Taken together, these results suggest an increased utility for TCCN spatial analysis in further refining the immune composition profiles that distinguish the TIME of responders and nonresponders beyond single-cell analysis alone.Given the intrapatient heterogeneity of HLA-I tumor expression observed at the single-cell level, we expanded the composition and spatial analysis of TCCN to account for the HLA-I status of the index tumor cell iTC; . GeneralDeep profiling of the TIME is providing new insights into the clinical heterogeneity of lymph node metastases as it relates to immunotherapeutic response. Single cell–based technologies have revolutionized the granularity by which the composition of the TIME can be probed . With thApplication of nearest neighborhood analysis to spatial mapping has been previously and extensively developed in the field of ecology, particularly in studies considering the spatial structures and diversity of forests . More reMultiplex imaging studies have predominantly focused on resolving clinical heterogeneity across a large patient cohort through analysis of tissue microarrays . BuildinAdvanced, multiplexed bioimaging platforms are undoubtedly enhancing our understanding of the spatial TIME but are not without limitations. Classification remains a significant challenge for many multiplex bioimaging modalities. Despite a high level of classification assignment to each sample and corresponding FOV, it is plausible that cell types that are not quantified in the abundance plot for a given sample are underrepresented. To address this potential shortcoming, the monocyte cell class was not evaluated in the downstream TCCN analysis, except for MPO-expressing neutrophils . In addition, further refinement of the antibody panel design in future studies, specifically the expansion of phenotypic markers and incorporation of functional markers including PDL1 will aid in refining classification for tumor and immune cell subsets. In our study, we selected patients with clear clinical response or failure to immunotherapy to consider two clinically distinct outcomes and the corresponding spatial landscapes. As such, our sample size was small and while insufficient to address sex-based differences, it was reflective of the clinical outcome experience as related to anti-PD1 immunotherapy treatment of stage IV melanoma . Future This study establishes MxIF as an effective platform to resolve patterns of paracrine interactions within the TIME of the metastatic melanoma lymph node as it relates to clinical response to PD1 immunotherapy. Through a comprehensive, multiplexed imaging approach, this work provides a framework for resolving spatial, cellular, and clinical heterogeneity in a biologically meaningful way utilizing the analytic unit of paracrine interaction, the TCCN. Our results demonstrate paracrine immune signatures within TCCNs are influenced by the anatomical region and HLA-I status of the iTC and refine the spatial landscape of the TIME beyond single-cell quantification alone. By demonstrating the viability of the MxIF INCell as a single-cell resolving biomaging platform, we have reaffirmed hypotheses surrounding the prognostic role of cytotoxic T cells and macrophages in the tumor core and identified new spatially relevant interactions between tumor and immune cell subsets with the potential to inform clinical responses to immunotherapy in the future.Supplementary Table 1Antibody reagents and validation for MxIF panel designClick here for additional data file.Supplementary Table 2Summary data of FOV distribution, single cells segmented, classified and clinical response to therapyClick here for additional data file.Supplementary Figure 1Phenotypic Cell Classification TreeClick here for additional data file.Supplementary Figure 2Virtual H&E images for whole excisional lymph node biopsies from each clinical caseClick here for additional data file.


Telomerase is a specialized reverse transcriptase that synthesizes telomeric repeats at the ends of linear chromosomes. Telomerase is transiently expressed in germ and stem cells, but nearly all somatic cells silence it after differentiating. However, the vast majority of cancer cells reactivate and constitutively express telomerase to maintain replicative immortality. Because of this, telomerase has remained a promising broad-spectrum chemotherapeutic target for over 30 years. However, various challenges associated with obtaining high-resolution structural data for telomerase have limited the development of rationally designed structure-based therapeutics. Various techniques and model systems have been utilized to advance our understanding of the structural biology of telomerase. In particular, multiple high-resolution cryogenic electron microscopy (cryo-EM) structures published within the past few years have revealed new components of the telomerase complex with near atomic resolution structural models. Additionally, these structures have provided details for how telomerase is recruited to telomeres and its mechanism of telomere synthesis. With these new pieces of evidence, and the promising outlook for future refinements to our current models, the possibility of telomerase specific chemotherapeutics is becoming more tangible than ever. This review summarizes these recent advancements and outlines outstanding questions in the field. Human t repeats .Human telomerase is a ribonucleoprotein (RNP) complex composed of the catalytic protein subunit telomerase reverse transcriptase (TERT), the telomerase RNA component (hTR), and an H/ACA RNP consisting of a single copy of TCAB1 and two copies each of dyskerin, GAR1, NHP2 and NOP10 ,16. ReceTetrahymena thermophila discovered a subdomain within the RT domain called TRAP, which forms an interface with the TEN domain to facilitate telomerase processivity by pinning hTR to TERT , the telomerase RNA binding domain (TRBD), the reverse transcriptase domain (RT), and the C-terminal extension (CTE) . Additio to TERT ,23. Subs to TERT  (23). Th to TERT  17). Th. ThTetra to TERT  24)..TetrahymTetrahymena thermophila's telomerase RNA component, which suggests the H2A–H2B interaction may be functionally similar  . Given tStructure-based drug design (SBDD) is a computational approach used to predict interactions between a drug candidate and its target protein. SBDD uses the three-dimensional structure of the target protein to design and optimize drug candidates that target specific sites on a protein to modulate its activity. This approach aims to minimize non-specific interactions between the drug and other macromolecules to maximize the effectiveness and potency of a drug candidate. Telomerase's heterogenous composition and configurational complexity, which had plagued early attempts to solve its structure, are finally beginning to be overcome thanks to technological improvements in cryo-EM structural determination. Continued improvements in data processing software will allow many of the heterogenous complexes to be resolved which may uncover new functional interactions and binding partners that have yet to be determined. However, the exact positions of amino acid sidechains and mobile nucleic acid elements may remain ambiguous without very high-resolution structures. Therefore, high resolution crystallographic or cryo-EM structures with well resolved sidechains are still required to characterize the specific molecular details of telomerase's activity, particularly within its active site during nucleotide addition. However, with near atomic resolution models, telomerase is finally becoming amenable to SBDD techniques.http://telomerase.asu.edu/). Mapping these mutations onto its structure reveals important interactions that may be targetable for telomerase inhibition  and artificial intelligence (AI). ML and AI’s ability to solve some of the most difficult problems in molecular biology has been demonstrated with the success of protein structure prediction programs such as AlphaFold . While MNo new data were generated or analysed in support of this research.zcad010_Supplemental_FilesClick here for additional data file.


Arrested cells also display an altered morphology of secretory organelles—engorgement of the trans-Golgi cisternae—that may reflect a disruption of post-Golgi secretory transport. We now ask whether trypanosomes under VSG silencing also reduce the rate of VSG turnover to further conserve coat density. Our data indicate that trypanosomes do not regulate VSG turnover according to VSG protein abundance, nor was there any effect on the post-Golgi transport of soluble or GPI-anchored secretory cargo. However, the surface morphology of silenced cells was altered from a typically rugose topology to a smoother profile, consistent with reduced overall membrane trafficking to the cell surface.African trypanosomes evade the immune system of the mammalian host by the antigenic variation of the predominant glycosylphosphatidylinositol (GPI)-anchored surface protein, variant surface glycoprotein (VSG). VSG is a very stable protein that is turned over from the cell surface with a long half-life (~26 h), allowing newly synthesized VSG to populate the surface. We have recently demonstrated that VSG turnover under normal growth is mediated by a combination of GPI hydrolysis and direct shedding with intact GPI anchors. VSG synthesis is tightly regulated in dividing trypanosomes, and when subjected to RNA interference (RNAi) silencing, cells display rapid cell cycle arrest in order to conserve VSG density on the cell surface  in a process called antigenic variation. VSG is essential, and when its synthesis is ablated by RNAi silencing, cells enter precytokinesis growth arrest as a means to maintain constant cell surface VSG levels. We have investigated whether arrested cells also alter the rate of natural VSG turnover as a means to conserve the surface coat. This work provides insights into the natural biology of the glycocalyx of this important human and veterinary parasite. Trypanosoma brucei spp. and other salivarian species) are the causative agents of human African trypanosomiasis (HAT) (African sleeping sickness) and the cattle wasting disease Nagana. Although HAT incidence has declined dramatically in the last decade (Glossina) and the mammalian host, where it can be found in the circulation, skin, adipose tissue, and central nervous system  of ~26 h -to-Golgi and then Golgi-to-lysosome trafficking are kinetically unimpaired  that were comparable to those previously observed in wild-type (WT) cells  cells . These rtrans-Golgi cisternae are dilated. The latter observation was interpreted to indicate that anterograde vesicular transport from the Golgi stacks is reduced in the absence of VSG cargo . Silencing eliminated this early BiP-associated VSG but had no effect on the transport and secretion of BiPN. Identical analyses were performed with the BiPNHP cell line (i). During intracellular trafficking, it was converted to the larger mature form (Nm) and was ultimately shed into the medium with typical kinetics. Silencing eliminated BiP-associated VSG at the initial time point but had no effect on reporter transport and shedding. These results indicate that the secretory arm of post-Golgi trafficking is not impaired by VSG silencing, as the transport and secretion/shedding of the matched reporters were unaffected.To test this, we constitutively expressed secretory reporters, soluble BiPN and GPI-anchored BiPNHP, in the VSG RNAi cell line. These matched reporters are based on the globular N-terminal ATPase domain of the ER molecular chaperone BiP. Soluble BiPN is transported to the flagellar pocket and then secreted to the external milieu , 16. BiPlar mass . These mlar mass . Endogenlar mass , was alsell line . Again, trans-Golgi cisternae  renders the parasite increasingly susceptible to innate arms of the host immune system, such as opsonization by the alternate complement pathway, leading to lysis and phagocytosis by macrophages. Second, VSG-depleted cells have markedly increased directional motility (less tumbling), which is attributed to increased cellular rigidity. We suggest that this loss of flexibility may be a natural consequence of the “contraction” of the total surface area that we observe by SEM. Finally, silenced cells have a reduced rate of clearance of surface-bound anti-VSG antibody. This was also attributed to the loss of flexibility and motility, but another contributing factor may be changes to the lipid composition of the plasma membrane due to reduced anterograde vesicular trafficking in the presence of ongoing membrane recycling/turnover.Our findings may shed light on three phenomena associated with the ablation of VSG synthesis, as reported previously by Cheung et al. . First, T. bruceibrucei (variant antigenic type MITat1.2 expressing VSG221) (2 (All work was carried out with cultured log-phase BSF cells of the commonly used tetracycline-responsive single-marker (SM) Lister 427 strain of  VSG221)  grown inG221) (2 . VSG RNAG221) (2  to lock G221) . RNA isolation, DNase treatment, cDNA synthesis, and quantitative PCR were performed as described previously , washed, resuspended in HMI9 medium with or without tetracycline, and incubated for the indicated times. Samples were lysed in radioimmunoprecipitation assay (RIPA) buffer , and labeled VSG 221 polypeptides were subsequently immunoprecipitated. Pulse and chase times are indicated in the figure legends. All immunoprecipitates were fractionated by 12% SDS-PAGE and imaged using a Molecular Dynamics Typhoon FLA 9000 system.Log-phase cultured BSF trypanosomes were RNAi silenced under tetracycline induction for 2 h prior to pulse-chase analysis. Following induction, cells were washed in HBS, and subsequent analysis was performed as described previously , with anThe secretory reporters BiPN (BiPN:AVRG ) and BiPScanning electron microscopy (SEM) analysis was performed as described previously . Brieflyhttp://imagej.nih.gov/ij/) was used to quantify data from phosphorimager assays and fluorescence blot scans. For quantitative analysis of band intensities, signals from each lane were subtracted from the signal of the equivalent unlabeled areas of that lane. In the case of the VSG turnover assay, the zero supernatant signal was subtracted from all subsequent supernatant signals. Data analyses were performed with Prism6 software . Biological replicates were obtained as indicated.ImageJ (