Single-cell RNA sequencing reveals peripheral blood leukocyte responses to spinal cord injury in mice with humanised immune systems.

Next-generation humanised mouse models and single-cell RNA sequencing (scRNAseq) approaches enable in-depth studies into human immune cell biology. Here we used NSG-SGM3 mice engrafted with human umbilical cord haematopoietic stem cells to investigate how human immune cells respond to and/or are changed by traumatic spinal cord injury (SCI). We hypothesised that the use of such mice could help advance our understanding of spinal cord injury-induced immune depression syndrome (SCI-IDS), and also how human leukocytes change as they migrate from the circulation into the lesion site. Our scRNAseq experiments, supplemented by flow cytometry, demonstrate the existence of up to 11 human immune cell (sub-) types and/or states across the blood and injured spinal cord (7 days post-SCI) of humanised NSG-SGM3 mice. Further comparisons of human immune cell transcriptomes between naïve, sham-operated and SCI mice identified a total of 579 differentially expressed genes, 190 of which were 'SCI-specific' (that is, genes regulated only in response to SCI but not sham surgery). Gene ontology analysis showed a prominent downregulation of immune cell function under SCI conditions, including for T cell receptor signalling and antigen presentation, confirming the presence of SCI-IDS and the transcriptional signature of human leukocytes in association with this phenomenon. We also highlight the activating influence of the local spinal cord lesion microenvironment by comparing the transcriptomes of circulating versus infiltrated human immune cells; those isolated from the lesion site were enriched for genes relating to both immune cell activity and function (e.g., oxidative phosphorylation, T cell proliferation and antigen presentation). We lastly applied an integrated bioinformatics approach to determine where immune responses in humanised NSG-SGM3 mice appear congruent to the native responses of human SCI patients, and where they diverge. Collectively, our study provides a valuable resource and methodological framework for the use of these mice in translational research.

Clinical validation of circulating GDF15/MIC-1 as a marker of response to docetaxel and survival in men with metastatic castration-resistant prostate cancer.

BACKGROUND: Elevated circulating growth differentiation factor (GDF15/MIC-1), interleukin 4 (IL4), and IL6 levels were associated with resistance to docetaxel in an exploratory cohort of men with metastatic castration-resistant prostate cancer (mCRPC). This study aimed to establish level 2 evidence of cytokine biomarker utility in mCRPC. METHODS: IntVal: Plasma samples at baseline (BL) and Day 21 docetaxel (n = 120). ExtVal: Serum samples at BL and Day 42 of docetaxel (n = 430). IL4, IL6, and GDF15 levels were measured by ELISA. Monocytes and dendritic cells were treated with 10% plasma from men with high or low GDF15 or recombinant GDF15. RESULTS: IntVal: Higher GDF15 levels at BL and Day 21 were associated with shorter overall survival (OS) (BL; p = 0.03 and Day 21; p = 0.004). IL4 and IL6 were not associated with outcomes. ExtVal: Higher GDF15 levels at BL and Day 42 predicted shorter OS (BL; p < 0.0001 and Day 42; p < 0.0001). Plasma from men with high GDF15 caused an increase in CD86 expression on monocytes (p = 0.03), but was not replicated by recombinant GDF15. CONCLUSIONS: Elevated circulating GDF15 is associated with poor prognosis in men with mCRPC receiving docetaxel and may be a marker of changes in the innate immune system in response to docetaxel resistance. These findings provide a strong rationale to consider GDF15 as a biomarker to guide a therapeutic trial of drugs targeting the innate immune system in combination with docetaxel in mCRPC.

Prophylactic Muscle Flaps Decrease Wound Complication Rates in Patients with Oncologic Spine Disease.

BACKGROUND: Patients with oncologic spine disease face a high systemic illness burden and often require surgical intervention to alleviate pain and maintain spine stability. Wound healing complications are the most common reason for reoperation in this population and are known to impact quality of life and initiation of adjuvant therapy. Prophylactic muscle flap (MF) closure is known to reduce wound healing complications in high-risk patients; however, the efficacy in oncologic spine patients is not well established. METHODS: A collaboration at our institution presented an opportunity to study the outcomes of prophylactic MF closure. The authors performed a retrospective cohort study of patients who underwent MF closure versus a cohort who underwent non-MF closure in the preceding time. Demographic and baseline health data were collected, as were postoperative wound complication data. RESULTS: A total of 166 patients were enrolled, including 83 patients in the MF cohort and 83 control patients. Patients in the MF group were more likely to smoke ( P = 0.005) and had a higher incidence of prior spine irradiation ( P = 0.002). Postoperatively, five patients (6%) in the MF group developed wound complications, compared with 14 patients (17%) in the control group ( P = 0.028). The most common overall complication was wound dehiscence requiring conservative therapy, which occurred in six control patients (7%) and one MF patient (1%) ( P = 0.053). CONCLUSIONS: Prophylactic MF closure during oncologic spine surgery significantly reduces the wound complication rate. Future studies should examine the precise patient population that stands to benefit most from this intervention. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Maximizing wound coverage in full-thickness skin defects: A randomized-controlled trial of autologous skin cell suspension and widely meshed autograft versus standard autografting.

BACKGROUND: Traumatic insults, infection, and surgical procedures can leave skin defects that are not amenable to primary closure. Split-thickness skin grafting (STSG) is frequently used to achieve closure of these wounds. Although effective, STSG can be associated with donor site morbidity, compounding the burden of illness in patients undergoing soft tissue reconstruction procedures. With an expansion ratio of 1:80, autologous skin cell suspension (ASCS) has been demonstrated to significantly decrease donor skin requirements compared with traditional STSG in burn injuries. We hypothesized that the clinical performance of ASCS would be similar for soft tissue reconstruction of nonburn wounds. METHODS: A multicenter, within-patient, evaluator-blinded, randomized-controlled trial was conducted of 65 patients with acute, nonthermal, full-thickness skin defects requiring autografting. For each patient, two treatment areas were randomly assigned to concurrently receive a predefined standard-of-care meshed STSG (control) or ASCS + more widely meshed STSG (ASCS+STSG). Coprimary endpoints were noninferiority of ASCS+STSG for complete treatment area closure by Week 8, and superiority for relative reduction in donor skin area. RESULTS: At 8 weeks, complete closure was observed for 58% of control areas compared with 65% of ASCS+STSG areas (p = 0.005), establishing noninferiority of ASCS+STSG. On average, 27.4% less donor skin was required with ASCS+ STSG, establishing superiority over control (p < 0.001). Clinical healing (≥95% reepithelialization) was achieved in 87% and 85% of Control and ASCS+STSG areas, respectively, at 8 weeks. The treatment approaches had similar long-term scarring outcomes and safety profiles, with no unanticipated events and no serious ASCS device-related events. CONCLUSION: ASCS+STSG represents a clinically effective and safe solution to reduce the amount of skin required to achieve definitive closure of full-thickness defects without compromising healing, scarring, or safety outcomes. This can lead to reduced donor site morbidity and potentially decreased cost associated with patient care.Clincaltrials.gov identifier: NCT04091672. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level I.

Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

BACKGROUND: Gallbladder cancer (GBC) is a prevalent and deadly biliary tract carcinoma, often diagnosed at advanced stages with limited treatment options. The 5-year survival rate varies widely from 4 to 60%, mainly due to differences in disease stage detection. With only a small fraction of patients having resectable tumors and a high incidence of metastasis, advanced GBC stages are characterized by significant chemoresistance. Identification of new therapeutic targets is crucial, and recent studies have shown that the Endothelin-1 (ET-1) signaling pathway, involving ETAR and/or ETBR receptors (ETRs), plays a crucial role in promoting tumor aggressiveness in various cancer models. Blocking one or both receptors has been reported to reduce invasiveness and chemoresistance in cancers like ovarian, prostate, and colon. Furthermore, transcriptomic studies have associated ET-1 levels with late stages of GBC; however, it remains unclear whether its signaling or its inhibition has implications for its aggressiveness. Although the role of ET-1 signaling in gallbladder physiology is minimally understood, its significance in other tumor models leads us to hypothesize its involvement in GBC malignancy. RESULTS: In this study, we investigated the expression of ET-1 pathway proteins in three GBC cell lines and a primary GBC culture. Our findings demonstrated that both ETAR and ETBR receptors are expressed in GBC cells and tumor samples. Moreover, we successfully down-regulated ET-1 signaling using a non-selective ETR antagonist, Macitentan, which resulted in reduced migratory and invasive capacities of GBC cells. Additionally, Macitentan treatment chemosensitized the cells to Gemcitabine, a commonly used therapy for GBC. CONCLUSION: For the first time, we reveal the role of the ET-1 pathway in GBC cells, providing insight into the potential therapeutic targeting of its receptors to mitigate invasion and chemoresistance in this cancer with limited treatment options. These findings pave the way for further exploration of Macitentan or other ETR antagonists as potential therapeutic strategies for GBC management. In summary, our study represents a groundbreaking contribution to the field by providing the first evidence of the ET 1 pathway's pivotal role in modulating the behavior and aggressiveness of GBC cells, shedding new light on potential therapeutic targets.

Cellular Context Dictates the Suppression or Augmentation of Triple-Negative Mammary Tumor Metastasis by NLRX1.

Prior studies have defined multiple, but inconsistent, roles for the enigmatic pattern recognition receptor NLRX1 in regulating several cancer-associated biological functions. In this study, we explore the role of NLRX1 in the highly metastatic murine 4T1 mammary tumor model. We describe a functional dichotomy of NLRX1 between two different cellular contexts: expression in healthy host cells versus expression in the 4T1 tumor cells. Using Nlrx1-/- mice engrafted with 4T1 tumors, we demonstrate that NLRX1 functions as a tumor suppressor when expressed in the host cells. Specifically, NLRX1 in healthy host cells attenuates tumor growth and lung metastasis through suppressing characteristics of epithelial-mesenchymal transition and the lung metastatic niche. Conversely, we demonstrate that NLRX1 functions as a tumor promoter when expressed in 4T1 tumor cells using gain- and loss-of-function studies both in vitro and in vivo. Mechanistically, NLRX1 in the tumor cells augments 4T1 aggressiveness and metastasis through regulating epithelial-mesenchymal transition hallmarks, cell death, proliferation, migration, reactive oxygen species levels, and mitochondrial respiration. Collectively, we provide critical insight into NLRX1 function and establish a dichotomous role of NLRX1 in the 4T1 murine mammary carcinoma model that is dictated by cellular context.

Measurable Residual Disease (MRD) by Flow Cytometry in Adult B-Acute Lymphoblastic Leukaemia (B-ALL) and Acute Myeloid Leukaemia (AML): Correlation with Molecular MRD Testing and Clinical Outcome at One Year.

Measurable residual disease (MRD) detected by flow cytometry (FC) is well established in paediatric B- lymphoblastic leukaemia (B-ALL) and adult chronic lymphocytic leukaemia (CLL), but its utility in adult B-ALL and adult acute myeloid leukaemia (AML) is less clear. In this prospective MRD study, one of the largest in Australia to date, we examined consecutive bone marrow aspirates from adult participants with B-ALL (n = 47) and AML (n = 87) sent for FC-MRD testing at a quaternary referral hospital in Sydney. FC-MRD results were correlated to corresponding Mol-MRD testing where available and clinical outcomes at three-month intervals over 1 year. B-ALL showed a moderate positive correlation (rs = 0.401, p < 0.001), while there was no correlation between FC-MRD and Mol-MRD for AML (rs = 0.13, p = 0.237). Five FC-MRD patterns were identified which had significant associations with relapse (X2(4) = 31.17(4), p > 0.001) and survival (X2(4) = 13.67, p = 0.008) in AML, but not in B-ALL. The three-month MRD results were also strongly associated with survival in AML, while the association in B-ALL was less evident. There was a moderate correlation between FC-MRD and Mol-MRD in B-ALL but not AML. The association of FC-MRD with relapse and survival was stronger in AML than in B-ALL. Overall, these findings suggest divergent utilities of FC-MRD in AML and B-ALL.

Enterothecal fistula as a rare cause of adult pneumocephalus and meningitis: a case report.

Background: Enterothecal fistulas are pathological connections between the gastrointestinal system and subarachnoid space. These rare fistulas occur mostly in pediatric patients with sacral developmental anomalies. They have yet to be characterized in an adult born without congenital developmental anomaly yet must remain on the differential diagnosis when all other causes of meningitis and pneumocephalus have been ruled out. Good outcomes rely on aggressive multidisciplinary medical and surgical care, which are reviewed in this manuscript. Case Description: A 25-year-old female with history of a sacral giant cell tumor resected via anterior transperitoneal approach followed by posterior L4-pelvis fusion presented with headaches and altered mental status. Imaging revealed that a portion of small bowel had migrated into her resection cavity and created an enterothecal fistula resulting in fecalith within the subarachnoid space and florid meningitis. The patient underwent a small bowel resection for fistula obliteration, and subsequently developed hydrocephalus requiring shunt placement and two suboccipital craniectomies for foramen magnum crowding. Ultimately, her wounds became infected requiring washouts and instrumentation removal. Despite a prolonged hospital course, she made significant recovery and at 10-month following presentation, she is awake, oriented, and able to participate in activities of daily living. Conclusions: This is the first case of meningitis secondary to enterothecal fistula in a patient without a previous congenital sacral anomaly. Operative intervention for fistula obliteration is the primary treatment and should be performed at a tertiary hospital with multidisciplinary capabilities. If recognized quickly and appropriately treated, there is a possibility of good neurological outcome.

A Consensus Approach for Targeted Muscle Reinnervation in Amputees.

Amputations have been performed with few modifications since the dawn of surgery. Blood vessels are ligated, bones are shortened, and nerves are cut. In a percentage of people, this can result in severe neuropathic, residual limb, and phantom limb pain. Targeted muscle reinnervation is a surgical procedure initially conceived to optimize function for myoelectric prostheses in amputees. Recently, it has been adopted more widely by surgeons for the prevention and treatment of neuropathic pain. Perhaps as a function of its relatively recent development, many authors perform this operation differently, and there has been no overall agreement regarding the principles, indications, technical specifics, and postoperative management guidelines. This article is written as a consensus statement by surgeons focused on the treatment of neuropathic pain and those with extensive experience performing targeted muscle reinnervation. It is designed to serve as a roadmap and template for extremity surgeons to consider when performing targeted muscle reinnervation.

An obstetrician-gynecologist's review of hernias: risk factors, diagnosis, prevention, and repair.

Management of obstetrical and gynecologic patients with hernias poses challenges to providers. Risks for hernia development include well-described factors that impair surgical wound healing and increase abdominal pressure. Among the diverse populations cared for by obstetricians and gynecologists, pregnant patients and those with gynecologic malignancies are at the highest risk for hernia formation. This article provides an overview of the existing literature, with a focus on patients cared for by obstetrician-gynecologists and commonly encountered preoperative and intraoperative scenarios. We highlight scenarios when a hernia repair is not commonly performed, including those of patients undergoing nonelective surgeries with known or suspected gynecologic cancers. Finally, we offer multidisciplinary recommendations on the timing of elective hernia repair with obstetrical and gynecologic procedures, with attention to the primary surgical procedure, the type of preexisting hernia, and patient characteristics.

Virtual Subinternships in the COVID Era: Lessons Learned from Three Institutional Experiences.

After the cessation of all in-person visiting rotations during the coronavirus 2019 pandemic, many programs developed virtual rotations as an alternative for the recruitment and education of prospective applicants. In this study, we developed a consortium of three institutions each with a unique virtual subinternship and prospectively surveyed participating students in order to reflect and improve upon future rotations. All students participating in virtual subinternships at three institutions were administered the same pre subinternship and post subinternship electronic surveys. Subinternship curricula were developed independently at each respective institution. Fifty-two students completed both surveys, for an overall response rate of 77.6%. Students' primary objectives were to evaluate their fit with the program (94.2%), interact with residents (94.2%), gain faculty mentorship (88.5%), and improve didactic knowledge (82.7%). Postrotation surveys revealed that over 73% of students reported having met all of these objectives over the course of the rotation. On average, students ranked programs 5% higher overall after the rotation (P = 0.024). Postrotation results showed that the majority (71.2%) of students perceived the virtual subinternship as slightly less valuable than in-person subinternships but that all students would participate in a virtual subinternship again. Student objectives can be successfully met using the virtual format for subinternships. The virtual format is also effective in enhancing the overall perception of a program and its residents. Although students still prefer in-person subinternships, our results suggest that virtual rotations are more accessible and very capable of meeting student goals.

Measurable residual disease in adult acute myeloid leukaemia: evaluation of a multidimensional 'radar' flow cytometric plot analysis method.

Measurable residual disease (MRD) monitoring in acute myeloid leukaemia (AML) is becoming increasingly important and is predominantly performed by multiparameter flow cytometry (MFC) or quantitative polymerase chain reactions (RT-qPCR). We investigated the use of multidimensional plots (MD-MFC) for AML MRD monitoring in an adult cohort. AML MRD was determined using a novel MD-MFC method for 115 MRD samples. Results were correlated with traditional two-dimensional MFC (2D-MFC) and molecular methods. Using the standard cut-off of 0.1% CD45+ cells, concordance was 99/115 (p=0.332). Eighty-four of 115 were concordant using a very low reporting limit of 0.01% (p=0.216). MRD <0.1% by either method was present in 40 of 115 samples. Fifteen of 40 were MD-MFC positive and 2D-MFC negative. Of these two of 15 had a molecular MRD marker and both were positive. Molecular MRD markers were available in 36 of 115 cases. Twenty-one of 36 (58%) were concordant with MD-MFC. Eight of 36 had detectable molecular MRD only and eight of 36 had positive MD-MFC only. There was no correlation between either the MFC method and the molecular results. In summary, there is good correlation between MD- and 2D-MFC-MRD and no correlation between the MFC and molecular methods.

The neuroprotective effects of estrogen and estrogenic compounds in spinal cord injury.

Spinal cord injury (SCI) occurs when the spinal cord is damaged from either a traumatic event or disease. SCI is characterised by multiple injury phases that affect the transmission of sensory and motor signals and lead to temporary or long-term functional deficits. There are few treatments for SCI. Estrogens and estrogenic compounds, however, may effectively mitigate the effects of SCI and therefore represent viable treatment options. This review systematically examines the pre-clinical literature on estrogen and estrogenic compound neuroprotection after SCI. Several estrogens were examined by the included studies: estrogen, estradiol benzoate, Premarin, isopsoralen, genistein, and selective estrogen receptor modulators. Across these pharmacotherapies, we find significant evidence that estrogens indeed offer protection against myriad pathophysiological effects of SCI and lead to improvements in functional outcomes, including locomotion. A STRING functional network analysis of proteins modulated by estrogen after SCI demonstrated that estrogen simultaneously upregulates known neuroprotective pathways, such as HIF-1, and downregulates pro-inflammatory pathways, including IL-17. These findings highlight the strong therapeutic potential of estrogen and estrogenic compounds after SCI.

Who's Auditioning Who? Applicant-Reported Elements of the Best and Worst Plastic Surgery Subinternships.

BACKGROUND: Many integrated plastic surgery applicants choose to complete one or more visiting subinternships or "away rotations" at programs outside of their home institution. As these rotations are so critical on both sides of the application process, the authors sought to identify the factors that influence subinternship experiences for plastic surgery applicants. METHODS: A survey was used to collect information about demographics, the subinternship experience, and interview preferences. The survey was distributed to current plastic surgery interns and applicants who applied to Duke Plastic Surgery in the 2019/2020 application cycle. RESULTS: One hundred forty-two responses were received (response rate, 35.2 percent). The mean number of subinternships completed was 4.47. The defining feature of respondents' best subinternship most often included engagement from faculty and residents, autonomy, and integration with the team. The worst feature of respondents' worst subinternship experience most often included a sense of disinterested or "malignant" residents and faculty, lacking operative/educational opportunities, and disorganization of the rotation. The majority of applicants (60.3 percent) would prefer to return for a standard interview day over interviewing while on rotation. CONCLUSIONS: The subinternship experience remains a critical part of the applicant experience when applying to integrated plastic surgery residency programs. The experience on these rotations leaves a lasting impression that is highly variable and influences future recommendations to peers. Rotating students value inclusivity and case volume, and they take note of negative interactions they witness among residents and faculty. These results can help as programs design their subinternship experience for visiting students in the future.

Muscle Flap Closures in Spine Surgery: Predictors of Usage Patterns and Factors Associated With Postoperative Complications From the NSQIP Database.

STUDY DESIGN: Retrospective cohort study using the National Surgical Quality Improvement Program. OBJECTIVE: The objective of this study was to identify preoperative factors that impact the decision to perform prophylactic muscle flap closure and assess risk factors for wound healing complications in patients undergoing spinal procedures with and without muscle flap closure. SUMMARY OF BACKGROUND DATA: Prior studies suggest that muscle flap closure following complex spine surgery results in a lower risk of wound healing complications. However, these studies have been limited to single institutions and/or surgeons. METHODS: The National Surgical Quality Improvement Program database was queried for all patients undergoing spine surgery between 2005 and 2017 with and without concomitant muscle flaps. Preoperative and perioperative variables were extracted. Univariate and multivariate analyses were performed to assess risk factors influencing surgical site infection (SSI) and wound disruption, as well as to delineate which preoperative factors increased the likelihood of patients receiving flap closures a priori. RESULTS: Concomitant muscle flaps were performed on 758 patients; 301,670 patients did not receive a flap. Overall 29 (3.83%) patients in the flap group experienced SSI compared to 5154 (1.71%) in the nonflap group (P<0.0001). Preoperative steroid use [odds ratio (OR) 0.5; P<0.0001], wound infection (OR 0.24; P<0.0001), elevated white blood cell count (OR 1.034; P<0.0001), low hematocrit (OR 0.94; P<0.0001), preoperative transfusion (OR 0.22; P=0.0068) were significantly associated with utilization of muscle flaps. Perioperative factors including a contaminated wound (OR 4.72; P<0.0001), the American Society of Anesthesiologists classification of severe disease (OR 1.92; P=0.024), and longer operative time (OR 1.001; P=0.0024) were significantly associated with postoperative wound disruption. In addition, after propensity score matching for these factors that increase risk of wound complications, there was no difference in the rates of SSI between the flap and nonflap group. CONCLUSION: Our results suggest that patients with a higher burden of illness preoperatively are more likely to receive prophylactic paraspinal flaps which can reduce the rates of wound-related complications.

Unravelling Checkpoint Inhibitor Associated Autoimmune Diabetes: From Bench to Bedside.

Immune checkpoint inhibitors have transformed the landscape of oncological therapy, but at the price of a new array of immune related adverse events. Among these is ß-cell failure, leading to checkpoint inhibitor-related autoimmune diabetes (CIADM) which entails substantial long-term morbidity. As our understanding of this novel disease grows, parallels and differences between CIADM and classic type 1 diabetes (T1D) may provide insights into the development of diabetes and identify novel potential therapeutic strategies. In this review, we outline the knowledge across the disciplines of endocrinology, oncology and immunology regarding the pathogenesis of CIADM and identify possible management strategies.

Description of the Nature of Musculoskeletal Shoulder Injuries in a Cohort of Commercial Truck Drivers: A Retrospective Cross-Sectional Study.

BACKGROUND: Commercial truck drivers (CTDs) are significantly affected by shoulder injuries; however, little is known about the unique mechanisms of injury (MOIs), specific injuries, or possible preventive measures among this group of workers. This study characterized the MOIs, musculoskeletal disorders (MSDs), and factors associated with MSDs of the shoulder among a group of CTDs. METHODS: A retrospective medical record review was conducted of CTDs between 21 and 65 years of age who were seen for MSDs of the shoulder between 2007 and 2015. RESULTS: A total of 130 CTDs were included, who were aged 21 to 65 years. Commercial truck drivers were most often injured during a fall (35%) or while using chains, tarps, or straps (31%). The two most common MSDs were unspecified sprains/strains (58%) and rotator cuff tears (24%). Age was found to be associated with all MSDs (p = .001) and an increased risk of developing rotator cuff tears (p =.005). Seventy-four percent of CTDs who experienced a rotator cuff tear were 46 years of age or older. CONCLUSION/APPLICATION TO PRACTICE: This study highlights the course of the injury in terms of diagnostics such as magnetic resonance imaging (MRI) and referral for surgery and describes the occupational activities associated with CTDs. These findings can inform employer injury prevention programs, patient and health care provider education, and future interventional research.

Complex I protein NDUFS2 is vital for growth, ROS generation, membrane integrity, apoptosis, and mitochondrial energetics.

Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.

The GDF15-GFRAL Pathway in Health and Metabolic Disease: Friend or Foe?

GDF15 is a cell activation and stress response cytokine of the glial cell line-derived neurotrophic factor family within the TGF-ß superfamily. It acts through a recently identified orphan member of the GFRα family called GFRAL and signals through the Ret coreceptor. Cell stress and disease lead to elevated GDF15 serum levels, causing anorexia, weight loss, and alterations to metabolism, largely by actions on regions of the hindbrain. These changes restore homeostasis and, in the case of obesity, cause a reduction in adiposity. In some diseases, such as advanced cancer, serum GDF15 levels can rise by as much as 10-100-fold, leading to an anorexia-cachexia syndrome, which is often fatal. This review discusses how GDF15 regulates appetite and metabolism, the role it plays in resistance to obesity, and how this impacts diseases such as diabetes, nonalcoholic fatty liver disease, and anorexia-cachexia syndrome. It also discusses potential therapeutic applications of targeting the GDF15-GFRAL pathway and lastly suggests some potential unifying hypotheses for its biological role.