BCL-2-family protein tBID can act as a BAX-like effector of apoptosis.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

Expanding roles of BCL-2 proteins in apoptosis execution and beyond.

The proteins of the BCL-2 family are known as key regulators of apoptosis, with interactions between family members determining permeabilisation of the mitochondrial outer membrane (MOM) and subsequent cell death. However, the exact mechanism through which they form the apoptotic pore responsible for MOM permeabilisation (MOMP), the structure and specific components of this pore, and what roles BCL-2 proteins play outside of directly regulating MOMP are incompletely understood. Owing to the link between apoptosis dysregulation and disease, the BCL-2 proteins are important targets for drug development. With the development and clinical use of drugs targeting BCL-2 proteins showing success in multiple haematological malignancies, enhancing the efficacy of these drugs, or indeed developing novel drugs targeting BCL-2 proteins is of great interest to treat cancer patients who have developed resistance or who suffer other disease types. Here, we review our current understanding of the molecular mechanism of MOMP, with a particular focus on recently discovered roles of BCL-2 proteins in apoptosis and beyond, and discuss what implications these functions might have in both healthy tissues and disease.

Abortion restrictions and medical residency applications.

Residency selection is a challenging process for medical students, one further complicated in the USA by the recent Dobbs v Jackson Women's Health Organization (Dobbs) decision over-ruling the federal right to abortion. We surveyed medical students to examine how Dobbs is influencing the ideological, personal and professional factors they must reconcile when choosing where and how to complete residency.Between 6 August and 22 October 2022, third-year and fourth-year US medical students applying to US residency programmes were surveyed through social media and direct outreach to medical schools. Analysis of quantitative and qualitative data from 494 responses was performed to assess downstream effects of Dobbs on residency applicants' family, health and career choices.Most respondents said changes in abortion access would likely or very likely influence their decision regarding location of considered residency programme (76.9%), where to start a family (72.2%) and contraceptive planning for them or their partner (57.9%). Cis-gender females were more influenced by Dobbs regarding where (5 (4, 5) p<0.001) and when (3 (3, 5) p<0.001) to start a family. In qualitative responses, medical trainees highlighted the importance of abortion access for their patients, themselves and their loved ones.Medical trainees are incorporating state abortion access into their residency programme choices. Future physicians care about both the quality of care they will be able to provide and their own health. For personal and professional reasons, reproductive healthcare access is now a key factor in residency match decisions.

Insemination "Fraud": Potentially Criminal Actions in Reproductive Medicine.

Case law and statutory provisions ensure marital rules of paternity apply when artificial insemination is associated with the pregnancy. Virtually all jurisdictions in the United States provide for gamete donors to remain anonymous. Much of this has been challenged with access to donor information via 23 and me. A breach of trust and a number of lawsuits involving physician provider(s) have resulted. We provide case law examples related to artificial insemination and the identification of the sperm donor. Proposed future legislation to protect patients and offspring from harm in relation to the process of donor sperm inseminations is provided.

Very Low Rates of Ureteral Injury in Laparoscopic Hysterectomy Performed by Fellowship-trained Minimally Invasive Gynecologic Surgeons.

STUDY OBJECTIVE: The objective of this case series is to evaluate the rates of ureteral injury at the time of laparoscopic hysterectomy among high-volume fellowship-trained surgeons. DESIGN: A retrospective chart review was performed, evaluating laparoscopic hysterectomy cases between 2009 and 2019 performed exclusively by fellowship-trained surgeons. SETTING: Division of Minimally Invasive Gynecologic Surgery (MIGS) at the Brigham and Women's Hospital and Brigham and Women's Faulkner Hospital, a Harvard Medical School teaching hospital in Boston. PATIENTS: All patients undergoing laparoscopic hysterectomy by one of 5 surgeons with fellowship training in MIGS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 5160 cases were handled by MIGS surgeons between 2009 and 2019 at our institution. Of these cases, 2345 were laparoscopic hysterectomy cases with available intraoperative and postoperative documentation. Most patients had undergone previous surgeries, and the most common indications for hysterectomy included uterine myomas, pelvic pain/endometriosis, and abnormal uterine bleeding. At the time of hysterectomy, 1 ureteral injury (0.04%) was noted. No additional delayed ureteral injuries were observed. Most patients were discharged home the same day (64.9%) and did not have any postoperative complications (63.9%) as designated by the Clavien-Dindo classification. CONCLUSION: Ureteral injury, although rare, is more prevalent in gynecologic surgery than in other surgical disciplines that have some focus on the pelvis. No study to date has evaluated the effect of surgical training and volume on rates of ureteral injuries. This study retrospectively examined ureteral injury rates for one group of high-volume fellowship-trained surgeons and found their rates to be lower than the national average. Proposals are presented for optimizing training and delivery of gynecologic surgical care to minimize complications.

Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization.

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

Spider toxin inhibits gating pore currents underlying periodic paralysis.

Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.

E2F1 interacts with BCL-xL and regulates its subcellular localization dynamics to trigger cell death.

E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes. This prevents efficient control of BCL-xL over its binding partners, in particular over BAK resulting in the induction of cell death. We thus identify a new, non-BH3-binding regulator of BCL-xL localization dynamics that influences its anti-apoptotic activity.

Impact of prior hysterectomy on surgical outcomes for laparoscopic adnexal surgery.

BACKGROUND: Adnexal surgery is believed to be more complex in patients with prior hysterectomy; however, there is little data regarding surgical outcomes. Understanding of individualized risks improves counseling, informed consent, and preoperative planning. METHODS: We performed a retrospective cohort study with a control group; we evaluated 744 patients undergoing laparoscopic adnexal surgery at an academic tertiary care center from 2011 to 2015. Comparisons were made using Chi square, Fisher's exact, or Wilcoxon-rank sum tests. We used log-binomial regression to calculate risk ratio and 95% confidence interval. RESULTS: Patients with prior hysterectomy were more likely to have intraoperative or postoperative complications at the time of laparoscopic adnexal surgery when compared to patients without prior hysterectomy [17.7% vs. 10.2%, p = 0.02, risk ratio (RR) 1.7, 95% confidence interval (CI) 1.1-2.7]. Patients with prior hysterectomy were four times more likely to have intraoperative complications (3.2% vs. 0.8%, p = 0.047, RR 4.0, 95% CI 1.1-14.7), and five times more likely to have conversion to laparotomy (5.6% vs. 1.1%, p = 0.004, RR 5.0, 95% CI 1.8-14.0). Patients with prior hysterectomy were more likely to need additional procedures, including lysis of adhesions (69.4% vs. 26.0%, p < 0.001), ureterolysis (15.3% vs. 4.8%, p < 0.001), and cystoscopy (28.2% vs. 8.1%, p < 0.001). They had longer operative time [101.5 min (IQR 59.5-135.0) vs. 78.0 min (IQR 53.0-109.0, p < 0.001)], and were less likely to have outpatient surgery (56.5% vs. 84.8%, p < 0.01). Postoperative complications were also more common (15.3% vs. 9.4%, p = 0.046). CONCLUSIONS: Patients with prior hysterectomy were 70% more likely to have a complication at the time of laparoscopic adnexal surgery than patients without hysterectomy. Increased risk of complications in subsequent adnexal surgery may influence the informed consent process or decisions regarding ovarian conservation. Awareness of potential need for additional surgical procedures may guide availability of equipment, choice of operating site, or referral to an advanced pelvic surgeon.

Surgeon Volume in Benign Gynecologic Surgery: Review of Outcomes, Impact on Training, and Ethical Contexts.

It is becoming increasingly clear that surgeon volume affects surgical outcomes. High-volume surgeons demonstrate reduced perioperative complications, shorter operative times, and reduced blood loss during multiple modalities of benign gynecologic surgery. Furthermore, high-volume surgeons consistently demonstrate higher rates of minimally invasive approaches, low rates of conversion to laparotomy, and lower per-procedure case costs. It is suggested that surgeons who have completed postresidency training have improved surgical outcomes, although these data are limited. Surgical exposure in obstetrics and gynecology residency is varied and does not consistently meet demonstrated surgical learning curves. Deficiencies in residency surgical training may be related to the volume-outcome relationship. We suggest reforming residency surgical training and tracking postresidency practice to provide optimal surgical care. Additionally, surgeons may have an ethical obligation to inform patients of their surgical volume and outcomes, with options for referrals if needed.

Whose Responsibility Is It to Define Exceptions in Abortion Bans?

This Viewpoint evaluates Texas' proposals to define the scope of the life exception for the state's abortion ban and argues that these approaches do not allow physicians to follow the national standards of care, avoid criminal liability, or have sufficient notice of what the law permits.

Structured vs narrative reporting of pelvic MRI for fibroids: clarity and impact on treatment planning.

OBJECTIVES: To evaluate clarity and usefulness of MRI reporting of uterine fibroids using a structured disease-specific template vs. narrative reporting for planning of fibroid treatment by gynaecologists and interventional radiologists. METHODS: This is a HIPAA-compliant, IRB-approved study with waiver of informed consent. A structured reporting template for fibroid MRIs was developed in collaboration between gynaecologists, interventional and diagnostic radiologists. The study population included 29 consecutive women who underwent myomectomy for fibroids and pelvic MRI prior to implementation of structured reporting, and 42 consecutive women with MRI after implementation of structured reporting. Subjective evaluation (on a scale of 1-10, 0 not helpful; 10 extremely helpful) and objective evaluation for the presence of 19 key features were performed. RESULTS: More key features were absent in the narrative reports 7.3 ± 2.5 (range 3-12) than in structured reports 1.2 ± 1.5 (range 1-7), (p < 0.0001). Compared to narrative reports, gynaecologists and radiologists deemed structured reports both more helpful for surgical planning (p < 0.0001) (gynaecologists: 8.5 ± 1.2 vs. 5.7 ± 2.2; radiologists: 9.6 ± 0.6 vs. 6.0 ± 2.9) and easier to understand (p < 0.0001) (gynaecologists: 8.9 ± 1.1 vs. 5.8 ± 1.9; radiologists: 9.4 ± 1.3 vs. 6.3 ± 1.8). CONCLUSION: Structured fibroid MRI reports miss fewer key features than narrative reports. Moreover, structured reports were described as more helpful for treatment planning and easier to understand. KEY POINTS: • Structured reports missed only 1.2 ± 1.5 out of 19 key features, as compared to narrative reports that missed 7.3 ± 2.5 key features for planning of fibroid treatment. • Structured reports were more helpful and easier to understand by clinicians. • Structured template can provide essential information for fibroids treatment planning.

Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem.

STUDY QUESTION: Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility? SUMMARY ANSWER: High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility. WHAT IS KNOWN ALREADY: There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform. STUDY DESIGN, SIZE, DURATION: Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 µM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase in fluorescence in the primary screen were rescreened and evaluated further, resulting in 48 hit compounds that produced a concentration-dependent increase in [Ca2+]i. Sperm penetration studies confirmed in vitro exposure to two hit compounds (A and B) resulted in significant improvement in functional motility in spermatozoa from healthy volunteer donors (A: 1 cm penetration index 2.54, 2 cm penetration index 2.49; P < 0.005 and B: 1 cm penetration index 2.1, 2 cm penetration index 2.6; P < 0.005), but crucially, also in patient samples from those undergoing fertility treatment (A: 1 cm penetration index 2.4; P = 0.009, 2 cm penetration index 3.6; P = 0.02 and B: 1 cm penetration index 2.2; P = 0.0004, 2 cm penetration index 3.6; P = 0.002). This was primarily as a result of direct or indirect CatSper channel action, supported by evidence from electrophysiology studies of individual sperm. LIMITATIONS, REASONS FOR CAUTION: Increase and fluxes in [Ca2+]i are fundamental to the regulation of sperm motility and function, including acrosome reaction. The use of calcium signalling as a surrogate for sperm motility is acknowledged as a potential limitation in this study. WIDER IMPLICATIONS OF THE FINDINGS: We conclude that HTS can robustly, efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoa and functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility. STUDY FUNDING/COMPETING INTEREST(S): The majority of the data were obtained using funding from TENOVUS Scotland and Chief Scientist Office NRS Fellowship. Additional funding was provided by NHS Tayside, MRC project grants (MR/K013343/1, MR/012492/1) and University of Abertay. The authors declare that there is no conflict of interest. TRAIL REGISTRATION NUMBER: N/A.

Fibroblast growth factor receptor 3 activation plays a causative role in urothelial cancer pathogenesis in cooperation with Pten loss in mice.

Although somatic mutations and overexpression of the tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) are strongly associated with bladder cancer, evidence for their functional involvement in the pathogenesis remains elusive. Previously we showed that activation of Fgfr3 alone is not sufficient to initiate urothelial tumourigenesis in mice. Here we hypothesize that cooperating mutations are required for Fgfr3-dependent tumourigenesis in the urothelium and analyse a mouse model in which an inhibitor of Pi3k-Akt signalling, Pten, is deleted in concert with Fgfr3 activation (UroIICreFgfr3(+/) (K644E) Pten(flox) (/flox)). Two main phenotypical characteristics were observed in the urothelium: increased urothelial thickness and abnormal cellular histopathology, including vacuolization, condensed cellular appearance, enlargement of cells and nuclei, and loss of polarity. These changes were not observed when either mutation was present individually. Expression patterns of known urothelial proteins indicated the abnormal cellular differentiation. Furthermore, quantitative analysis showed that Fgfr3 and Pten mutations cooperatively caused cellular enlargement, while Pten contributed to increased cell proliferation. Finally, FGFR3 overexpression was analysed along the level of phosphorylated mTOR in 66 T1 urothelial tumours in tissue microarray, which supported the occurrence of functional association of these two signalling pathways in urothelial pathogenesis. Taken together, this study provides evidence supporting a functional role of FGFR3 in the process of pathogenesis in urothelial neoplasms. Given the wide availability of inhibitors specific to FGF signalling pathways, our model may open the avenue for FGFR3-targeted translation in urothelial disease.

Maternal Cardiovascular Health Post-Dobbs.

Maternal Cardiovascular Health Post-DobbsPregnancy is associated with increasing morbidity and mortality in the United States. In the post-Dobbs era, many pregnant patients at highest risk no longer have access to abortion, which has been a crucial component of standard medical care.

Modulation of ribosomal frameshifting frequency and its effect on the replication of Rous sarcoma virus.

Programmed -1 ribosomal frameshifting is widely used in the expression of RNA virus replicases and represents a potential target for antiviral intervention. There is interest in determining the extent to which frameshifting efficiency can be modulated before virus replication is compromised, and we have addressed this question using the alpharetrovirus Rous sarcoma virus (RSV) as a model system. In RSV, frameshifting is essential in the production of the Gag-Pol polyprotein from the overlapping gag and pol coding sequences. The frameshift signal is composed of two elements, a heptanucleotide slippery sequence and, just downstream, a stimulatory RNA structure that has been proposed to be an RNA pseudoknot. Point mutations were introduced into the frameshift signal of an infectious RSV clone, and virus replication was monitored following transfection and subsequent infection of susceptible cells. The introduced mutations were designed to generate a range of frameshifting efficiencies, yet with minimal impact on encoded amino acids. Our results reveal that point mutations leading to a 3-fold decrease in frameshifting efficiency noticeably reduce virus replication and that further reduction is severely inhibitory. In contrast, a 3-fold stimulation of frameshifting is well tolerated. These observations suggest that small-molecule inhibitors of frameshifting are likely to have potential as agents for antiviral intervention. During the course of this work, we were able to confirm, for the first time in vivo, that the RSV stimulatory RNA is indeed an RNA pseudoknot but that the pseudoknot per se is not absolutely required for virus viability.

The role of the robot in treating urinary tract endometriosis.

PURPOSE OF REVIEW: To detail the recent advances in the use of computer-enhanced robotic technology to surgically treat urinary tract endometriosis. RECENT FINDINGS: Few studies have been published in this field. The studies are severely limited in scope. Further study is warranted. SUMMARY: Robotic-assisted laparoscopic techniques have proven useful in the treatment of extensive endometriosis and may prove useful in the treatment of urinary tract endometriosis.