Bladder Stones in Renal Transplant Patients: Presentation, Management, and Follow-up.

INTRODUCTION: The study aim was to analyze the presentation, management, and follow-up of renal transplant patients developing bladder calculi. METHODS: Patients who underwent renal transplant with postoperative follow-up at our institution were retrospectively analyzed (1984-2023) to assess for the development of posttransplant bladder stones. All bladder stones were identified by computerized tomography imaging and stone size was measured using this imaging modality. RESULTS: The prevalence of bladder calculi post-renal transplantation during the study window was 0.22% (N = 20/8,835) with a median time to bladder stone diagnosis of 13 years posttransplant. Of all bladder stone patients, 6 (30%) received deceased donor and 14 (70%) living donor transplants. There were 11 patients with known bladder stone composition available; the most common being calcium oxalate (N = 6). Eleven (55%) patients had clinical signs or symptoms (most commonly microhematuria). Fourteen of the bladder stone cohort patients (70%) underwent treatment including cystolitholapaxy in 12 subjects. Of these 14 patients, 9 (64%) were found to have nonabsorbable suture used for their ureteroneocystostomy closure. CONCLUSIONS: The prevalence of bladder stones post-renal transplant is low. The utilization of nonabsorbable suture for ureteral implantation was the main risk factor identified in our series. This technique is no longer used at our institution. Other factors contributing to bladder stone formation in this population warrant identification.

Single center experience and literature review of kidney transplantation from non-ideal donors with acute kidney injury: Risk and reward.

INTRODUCTION: There is limited experience transplanting kidneys from either expanded criteria donors (ECD) or donation after circulatory death (DCD) deceased donors with terminal acute kidney injury (AKI). METHODS: AKI kidneys were defined by a donor terminal serum creatinine level >2.0 mg/dL whereas non-ideal deceased donor (NIDD) kidneys were defined as AKI/DCD or AKI/ECDs. RESULTS: From February 2007 to March 2023, we transplanted 266 single AKI donor kidneys including 29 from ECDs, 29 from DCDs (n = 58 NIDDs), and 208 from brain-dead standard criteria donors (SCDs). Mean donor age (43.7 NIDD vs. 33.5 years SCD), KDPI (66% NIDD vs. 45% SCD), and recipient age (57 NIDD vs. 51 years SCD) were higher in the NIDD group (all p < .01). Mean waiting times (17.8 NIDD vs. 24.2 months SCD) and dialysis duration (34 NIDD vs. 47 months SCD) were shorter in the NIDD group (p < .05). Delayed graft function (DGF, 48%) and 1-year graft survival (92.7% NIDD vs. 95.9% SCD) was similar in both groups. Five-year patient and kidney graft survival rates were 82.1% versus 89.9% and 82.1% versus 75.2% (both p = NS) in the NIDD versus SCD groups, respectively. CONCLUSIONS: The use of kidneys from AKI donors can be safely liberalized to include selected ECD and DCD donors.

Schizosaccharomyces pombe Ccq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase-telomere association.

The telomerase protein Est1 exists in multiple organisms, including Schizosaccharomyces pombe, humans, and Saccharomyces cerevisiae, but its function has only been closely examined in S. cerevisiae, where it is a recruiter/activator of telomerase. Here, we demonstrate that S. pombe Est1 was required for the telomere association of the telomerase holoenzyme, suggesting that it too has a recruitment role. Its association with telomeres was dependent on Trt1, the catalytic subunit, and Ccq1, a telomeric protein. Surprisingly, Est1 telomere binding was only partially dependent on TER1, the telomerase RNA, even though Est1 bound nucleotides 415-507 of TER1. A ter1-Δ415-507 strain had short telomeres and very low Est1 and Trt1 telomere association in late S phase but did not senesce. An unbiased search for mutations that reduced Est1-TER1 interaction identified mutations only in the Est1 14-3-3-like domain, a phosphoserine-binding motif, the first example of a 14-3-3-like domain with RNA-binding activity. These mutations also reduced Est1-Ccq1 binding. One such mutant prevented Est1 telomere association and caused telomere loss and slow senescence, similar to ccq1Δ. We propose that the Est1-Ccq1 interaction is critical for telomerase recruitment, while the Est1-TER1 interaction acts downstream from Ccq1-mediated recruitment to stabilize the holoenzyme at the telomere.

DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase.

Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

A CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S. cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.

Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding.

The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

Telomerase RNA is more than a DNA template.

The addition of telomeric DNA to chromosome ends is an essential cellular activity that compensates for the loss of genomic DNA that is due to the inability of the conventional DNA replication apparatus to duplicate the entire chromosome. The telomerase reverse transcriptase and its associated RNA bind to the very end of the telomere via a sequence in the RNA and specific protein-protein interactions. Telomerase RNA also provides the template for addition of new telomeric repeats by the reverse-transcriptase protein subunit. In addition to the template, there are 3 other conserved regions in telomerase RNA that are essential for normal telomerase activity. Here we briefly review the conserved core regions of telomerase RNA and then focus on a recent study in fission yeast that determined the function of another conserved region in telomerase RNA called the Stem Terminus Element (STE). (1) The STE is distant from the templating core of telomerase in both the linear and RNA secondary structure, but, nonetheless, affects the fidelity of telomere sequence addition and, in turn, the ability of telomere binding proteins to bind and protect chromosome ends. We will discuss possible mechanisms of STE action and the suitability of the STE as an anti-cancer target.

Impact of endoscopic stapling of Zenker's diverticulum on patient health status as assessed by the Glasgow Benefit Inventory.

OBJECTIVES: The aim of this study was to measure the effect of endoscopic stapling of Zenker's diverticulum on the health status of patients. METHODS: We used the Glasgow Benefit Inventory (GBI) tool to ascertain patients' perceived experience. RESULTS: The study demonstrated a positive health impact following endoscopic stapling of the Zenker's diverticulum. The mean total GBI score was +18.9 (95% confidence interval, +/- 11.2). The impact of endoscopic stapling was positive for all three subsets of the GBI. The GBI scores were comparable to those obtained after other common otorhinolaryngological procedures such as tonsillectomy, rhinoplasty, and middle ear surgery. CONCLUSIONS: These data should improve our ability to counsel patients regarding important therapeutic decisions and expectations of surgical outcome.

Endoscopic stapling of Zenker's diverticulum: establishing national baselines for auditing clinical outcomes in the United Kingdom.

Endoscopic stapling of Zenker's diverticulum (ZD) is now established practice in the UK and is routinely performed by the vast majority of otolaryngologists. Both The National Confidential Enquiry into Peri-Operative Deaths and the National Institute for Health and Clinical Excellence recommended that the procedure be undertaken at specialist centres and that each department should audit their respective outcomes. Despite the abundance of review articles, it remains unclear what variables a meaningful audit is required to collate and what gold standards every department in the UK undertaking surgery for ZD should aspire to achieve. The objective was to review the outcomes of endoscopic stapling of Zenker's diverticulum at this institution. In addition, a review of other UK departments was undertaken to formulate minimum clinical standards and recommendations of best practice. Review of patient case notes and a structured search of PubMed were used as materials. Fifteen retrospective case series were identified from the literature search which fulfilled the inclusion criteria. Fifty-one patients were identified from the present audit. When these were added to the review, a total of 585 patients were available for meta-analysis. 540 (92.3%) were successfully stapled. Forty-five (7.7%) procedures were abandoned intra-operatively. The most common reason was difficulty assessing the small pouch. The majority of patients (92%) had resumed oral intake by the second post-operative day. Most patients (87%) were discharged by the second post-operative day. Outcomes were good with over 90% reporting resolved or significantly improved symptoms. Minor complications included dental trauma, transient hoarseness and sore throat causing delayed resumption of oral intake. The overall perforation rate was 4.8%. One death was reported. In conclusion endoscopic stapling of pharyngeal pouch is a safe procedure that is associated with good outcomes and low complication rates. No death has been reported since 2000 in the UK. The data presented in this review represent current clinical standards reported over the past 15 years by UK departments and serve as a useful benchmark for any future audits undertaken.

Successful Treatment of Hard Corns in Two Patients Using Microwave Energy.

Corns are a common foot problem accounting for nearly half of all problems seen within podiatry and chiropody clinics. Hard corns are concentrated areas of hyperkeratosis within the stratum corneum, typically found on the weight-bearing (plantar) surfaces of the feet. For many patients, they are a source of pain and have been shown to negatively affect a patient's activity and quality of life. Most of the currently available treatments are short-lived in their effectiveness, with corns frequently being recurrent, requiring repeated visits to remove the painful lesions. The use of handheld microwave devices indicated for surface applications has demonstrated effectiveness in clearing recalcitrant plantar warts and significantly reducing the pain associated with them. The authors report 2 cases of patients with persistent and painful plantar corns who underwent microwave treatment of their plantar corns with a successful, lasting reduction in pain levels after the intervention. Further work is required to fully assess the potential of this treatment modality in the management of painful plantar corns.

Is There an Upper Age Limit for Bariatric Surgery? Laparoscopic Gastric Bypass Outcomes in Septuagenarians.

PURPOSE: Upper age limits for bariatric surgery are questioned on the merits of increased complication rates in the elderly and questionable efficacy. This study evaluates outcomes of bariatric surgery in patients ≥ 70 years of age. MATERIALS AND METHODS: Retrospective review was performed of patients ≥ 70 years of age who underwent laparoscopic Roux-en-Y gastric bypass (RYGB) between 2001 and 2018. Primary endpoints were 30-day readmission, Clavien-Dindo grade III-V (CD III-IV) complications, and mortality. Secondary data included were weight loss, long-term outcomes, comorbidity resolution, hemoglobin A1C, and lipid panels. RESULTS: A total of 23 patients with an average age of 72 years (range 70-80 years) and mean BMI of 43.3 (range 37.3-56.0) were reviewed. Average length-of-stay was 2.4 days (range 1-6 days), with the only acute complication being aspiration pneumonia in one patient. Median follow-up was 69.3 weeks (range 9-875 weeks). One-year follow-up rate was 96%, during which no deaths or CD III-IV complications occurred. Subsequently, one patient experienced failure-to-thrive requiring temporary enteral nutrition. Average 1 year percent total weight loss (%TWL) was 29%, and this was maintained on subsequent follow-ups. Average 1 year percent excess weight loss (%EWL) was 60%, maintained long-term at 61%. Significant serum biochemical improvements included hemoglobin A1C (6.9 ± 1.4% to 5.6 ± 1.3%, p = 0.001), triglycerides (155 ± 49 mg/dL to 102 ± 41 mg/dL, p = 0.0003), and high-density lipoprotein cholesterol (48 ± 14 mg/dL to 58 ± 22 mg/dL, p = 0.004). CONCLUSION: Laparoscopic RYGB is a safe and effective treatment for obesity and obesity-related comorbidities in septuagenarians.

Analysis of mutant phenotypes and splicing defects demonstrates functional collaboration between the large and small subunits of the essential splicing factor U2AF in vivo.

The heterodimeric splicing factor U2AF plays an important role in 3' splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3' splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3' splice site recognition, we sought introns for which the spU2AFLG and spU2AFSM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract. These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.

The splicing factor U2AF small subunit is functionally conserved between fission yeast and humans.

The small subunit of U2AF, which functions in 3' splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AF(SM)) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AF(SM) depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.

A simple and reliable predictor for an adequate laryngeal view with rigid endoscopic laryngoscopy.

OBJECTIVES: It is sometimes impossible to obtain an adequate laryngeal view during rigid endoscopic laryngoscopy. This may be due to a high tongue base. Our study seeks to determine a correlation between tongue base level and the adequacy of laryngeal view obtained with a 70-degree rigid endoscope. STUDY DESIGN AND SETTING: Over a period of 4 months, patients from a voice clinic were gathered and categorized into class I to III according to Mallampati et al (1985). Rigid laryngo-videostroboscopy was conducted to assess the larynx and the adequacy of the view was recorded. RESULTS: 74 patients were recruited. The number of adequate views were: class I = 18/20 (90%); class II = 20/33 (60.6%); class III = 7/21 (33.3%). chi 2 analysis demonstrated significance trend in all 3 classes. CONCLUSION: The level of the tongue base correlated well with the adequacy of laryngeal view obtained from a 70-degree rigid endoscope. This can be used to predict the success of obtaining adequate views during rigid laryngoscopy.

The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening.

Pif1 family helicases are evolutionary conserved 5'-3' DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.

DNA repair at telomeres: keeping the ends intact.

The molecular era of telomere biology began with the discovery that telomeres usually consist of G-rich simple repeats and end with 3' single-stranded tails. Enormous progress has been made in identifying the mechanisms that maintain and replenish telomeric DNA and the proteins that protect them from degradation, fusions, and checkpoint activation. Although telomeres in different organisms (or even in the same organism under different conditions) are maintained by different mechanisms, the disparate processes have the common goals of repairing defects caused by semiconservative replication through G-rich DNA, countering the shortening caused by incomplete replication, and postreplication regeneration of G tails. In addition, standard DNA repair mechanisms must be suppressed or modified at telomeres to prevent their being recognized and processed as DNA double-strand breaks. Here, we discuss the players and processes that maintain and regenerate telomere structure.

Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA.

Although the catalytic subunit of the Schizosaccharomyces pombe telomerase holoenzyme was identified over ten years ago, the unusual heterogeneity of its telomeric DNA made it difficult to identify its RNA component. We used a new two-step immunoprecipitation and reverse transcription-PCR technique to identify the S. pombe telomerase RNA, which we call TER1. TER1 RNA was 1,213 nucleotides long, similar in size to the Saccharomyces cerevisiae telomerase RNA, TLC1. TER1 RNA associated in vivo with the two known subunits of the S. pombe telomerase holoenzyme, Est1p and Trt1p, and neither association was dependent on the other holoenzyme component. We present a model to explain how telomerase introduces heterogeneity into S. pombe telomeres. The technique used here to identify TER1 should be generally applicable to other model organisms.

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin.

Discrete sequence elements known as exonic splicing enhancers (ESEs) have been shown to influence both the efficiency of splicing and the profile of mature mRNAs in multicellular eukaryotes. While the existence of ESEs has not been demonstrated previously in unicellular eukaryotes, the factors known to recognize these elements and mediate their communication with the core splicing machinery are conserved and essential in the fission yeast Schizosaccharomyces pombe. Here, we provide evidence that ESE function is conserved through evolution by demonstrating that three exonic splicing enhancers derived from vertebrates (chicken ASLV, mouse IgM, and human cTNT) promote splicing of two distinct S. pombe pre-messenger RNAs (pre-mRNAs). Second, as in extracts from mammalian cells, ESE function in S. pombe is compromised by mutations and increased distance from the 3'-splice site. Third, three-hybrid analyses indicate that the essential SR (serine/arginine-rich) protein Srp2p, but not the dispensable Srp1p, binds specifically to both native and heterologous purine-rich elements; thus, Srp2p is the likely mediator of ESE function in fission yeast. Finally, we have identified five natural purine-rich elements from S. pombe that promote splicing of our reporter pre-mRNAs. Taken together, these results provide strong evidence that the genesis of ESE-mediated splicing occurred early in eukaryotic evolution.