Unilateral keratitis secondary to Leishmania spp. infection in a horse: Clinical signs and successful topical therapy.

Dermatological clinical signs have been seldom reported in the literature secondary to equine leishmaniasis. This case depicts the clinical signs, treatment, and outcome of a young horse with a pink, elevated lesion on the ventromedial quadrant of the cornea. A corneal cytology was performed and revealed the presence of leishmania amastigotes reaching the diagnosis of keratitis secondary to leishmania. Surgical resection was recommended but the owner declined the procedure, and the lesion was treated with a topical antimonial for 6 weeks. The lesion reduced remarkably during the first weeks of treatment. The patient had not shown recurrence of the lesion for 2 years since the treatment was started. Leishmania spp. can be responsible for ocular surface abnormalities such as keratitis. Corneal cytology is an inexpensive diagnostic method that should be considered when ocular surface abnormalities are identified in horses in endemic areas.

Single-chain dimers from de novo immunoglobulins as robust scaffolds for multiple binding loops.

Antibody derivatives have sought to recapitulate the antigen binding properties of antibodies, but with improved biophysical attributes convenient for therapeutic, diagnostic and research applications. However, their success has been limited by the naturally occurring structure of the immunoglobulin dimer displaying hypervariable binding loops, which is hard to modify by traditional engineering approaches. Here, we devise geometrical principles for de novo designing single-chain immunoglobulin dimers, as a tunable two-domain architecture that optimizes biophysical properties through more favorable dimer interfaces. Guided by these principles, we computationally designed protein scaffolds that were hyperstable, structurally accurate and robust for accommodating multiple functional loops, both individually and in combination, as confirmed through biochemical assays and X-ray crystallography. We showcase the modularity of this architecture by deep-learning-based diversification, opening up the possibility for tailoring the number, positioning, and relative orientation of ligand-binding loops targeting one or two distal epitopes. Our results provide a route to custom-design robust protein scaffolds for harboring multiple functional loops.

Individually adjusted absolute blood volume feedback control: A promising solution for intradialytic hypotension.

INTRODUCTION: Intradialytic hypotension (IDH) remains one of the most frequent complications associated to hemodialysis (HD), frequently triggered by a reduction in absolute blood volume (ABV) not compensated by vascular refilling. A recently developed dilutional method allows routinary measurement of ABV and, by a simple algorithm, may turn blood volume monitor (BVM) guided UF (ultrafiltration) biofeedback into an ABV control, automatically adjusting UF rate to maintain ABV above a preset threshold. The aim of this study is to identify an individual critical ABV threshold and test the ability of an ABV feedback control to avoid IDH. METHODS: We studied 24 patients throughout three consecutive midweek HD treatments. ABV and blood pressure (BP) were measured every 30 min and anytime the patient referred any symptoms to identify each patient's critical ABV (ABV at the time of hypotension). A fixed bolus dilution approach at the start of HD was used to calculate ABV. Then, patients were followed through three additional HD treatments and IDH development was analyzed. FINDINGS: Seventy-one treatments performed in 24 patients. ABV monitoring showed a constant decrease as HD treatment progressed. Thirteen IDH events were observed in eight different patients, with a mean systolic BP drop in IDH treatments of 37.38 ± 4.31 mmHg and a mean adjusted ABV at hypotension of 71.07 ± 14.88 mL/kg. Critical ABV was individually set in patients prone to IDH. As expected, ABV feedback control successfully maintained ABV over preset critical ABV. IDH events were avoided in 21 out of 22 treatments performed. ABV drop was successfully reduced, as well as SBP drop (despite similar UF than prior to ABV feedback control implementation). DISCUSSION: ABV feedback control avoided IDH in 21 out of 22 treatments performed by maintaining blood volume above critical ABV, significantly reducing ABV variations without compromising prescribed UF.

Structure and function analysis of Sam68 and hnRNP A1 synergy in the exclusion of exon 7 from SMN2 transcripts.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the absence of a functional copy of the Survival of Motor Neuron 1 gene (SMN1). The nearly identical paralog, SMN2, cannot compensate for the loss of SMN1 because exon 7 is aberrantly skipped from most SMN2 transcripts, a process mediated by synergistic activities of Src-associated during mitosis, 68 kDa (Sam68/KHDRBS1) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1. This results in the production of a truncated, nonfunctional protein that is rapidly degraded. Here, we present several crystal structures of Sam68 RNA-binding domain (RBD). Sam68-RBD forms stable symmetric homodimers by antiparallel association of helices α3 from two monomers. However, the details of domain organization and the dimerization interface differ significantly from previously characterized homologs. We demonstrate that Sam68 and hnRNP A1 can simultaneously bind proximal motifs within the central region of SMN2 (ex7). Furthermore, we show that the RNA-binding pockets of the two proteins are close to each other in their heterodimeric complex and identify contact residues using crosslinking-mass spectrometry. We present a model of the ternary Sam68·SMN2 (ex7)·hnRNP A1 complex that reconciles all available information on SMN1/2 splicing. Our findings have important implications for the etiology of SMA and open new avenues for the design of novel therapeutics to treat splicing diseases.

De novo design of immunoglobulin-like domains.

Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) ß-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture-the non-local cross-ß structure connecting the two ß-sheets-and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties.

Impact of dialysate sodium concentration on vascular refilling.

BACKGROUND: Although relationship between dialysate sodium concentration and hemodynamic stability has been well studied over the years, outcomes of absolute blood volume (ABV) maintenance and vascular refilling volume (Vref ) modifications were not included, as its analysis has not been easily accessible to direct investigation. However, recent studies report a simple and feasible methodology to assess ABV and Vref during hemodialysis (HD) treatments. It is the aim of this study to analyze whether sodium concentration in dialysate modifies ABV drop and Vref . METHODS: The study was performed in 19 patients under HD. During three different sessions, sodium concentration in dialysate was randomized to three different profiles: low sodium concentration (LNa, 138 mEq/L), neutral sodium concentration (NNa, 140 mEq/L), and high sodium concentration (HNa, 143 mEq/L). ABV and Vref were calculated using Kron et al methodology. RESULTS: Predialysis values of the measured parameters showed similar results for the three profiles. Sodium concentration showed an effect on ABV drop, Vref, and vascular refilling fraction (Fref ). Pair-wise comparison revealed mean ABV decreased 0.21 L less when using HNa profile versus LNa profile (p = 0.027), a mean Vref increase of 0.39 L (p = 0.038), and a mean Fref increase of 9.94% (p = 0.048). CONCLUSIONS: This study shows that the use of HNa profiles increases Vref and Fref and reduces ABV drop during dialysis treatments when compared to LNa profiles.

Effect of Beta-Blocker Cardioselectivity on Vascular Refilling in Hemodialysis Patients.

BACKGROUND: ß-Blockers are the most frequently prescribed cardioprotective drugs in hemodialysis (HD) patients, despite their weak evidence. We sought to evaluate the effects of ß-blockers on vascular refilling during HD treatments and examine whether carvedilol, for being noncardioselective and poorly dialyzable, associates more impact than others. METHODS: The study was performed in a cohort of maintenance HD patients from a tertiary center. All patients had previous ß-blocker prescription. We conducted a prospective crossover study and measured vascular refilling volume (Vref) and vascular refilling fraction (Fref) in 2 circumstances: under ß-blocker treatment (ßb profile) and without ß-blocker effect (non-ßb profile). RESULTS: Twenty patients were included, 10 of whom were treated with carvedilol. Predialysis values were comparable between the 2 profiles. Although the ßb profile showed lower Vref and higher ABV drop, these differences did not reach statistical significance. Data showed an increase in Fref in the non-ßb profile (70.01 ± 6.80% vs. 63.14 ± 11.65%; p = 0.015). The ßb profile associated a significantly higher risk of intradialytic hypotension (IDH) (risk ratio 2.40; 95% CI: 1.04-5.55). When analyzing separately the carvedilol group, patients dialyzed under drug effect experienced a significant impairment in Vref, Fref, and refilling rate. CONCLUSIONS: Administering ß-blockers before HD associated a higher risk of IDH and a decrease in Fref. Patients dialyzed under carvedilol effect showed an impaired refilling, probably related to its noncardioselectivity and lower dializability.

Safety of renal biopsy bleeding prophylaxis with desmopressin.

BACKGROUND: Percutaneous renal biopsy (PRB) is invasive, and bleeding-related complications are a concern. Desmopressin (DDAVP) is a selective type 2 vasopressin receptor-agonist also used for haemostasis. AIM: To evaluate the side effects of intravenous (IV) weight-adjusted desmopressin preceding PRB. METHODS: This was a retrospective study of renal biopsies performed by nephrologists from 2013 to 2017 in patients who received single-dose DDAVP pre-PRB. RESULTS: Of 482 PRBs, 65 (13.5%) received DDAVP (0.3 µg/kg); 55.4% of the PRBs were native kidneys. Desmopressin indications were altered platelet function analyser (PFA)-100 results (75.3% of the patients), urea >24.9 mmol/L (15.5%), antiplatelet drugs (6.1%) and thrombocytopaenia (3%). Of the 65 patients, 30.7% had minor asymptomatic complications, and 3 patients had major complications. Pre-PRB haemoglobin (Hb) <100 g/L was a risk factor for Hb decrease >10 g/L, and altered collagen-epinephrine (Col-Epi) time was a significant risk factor for overall complications. Mean sodium decrease was 0.6 ± 3 mmol/L. Hyponatraemia without neurological symptoms was diagnosed in two patients; no cardiovascular events occurred. CONCLUSION: Hyponatraemia after single-dose DDAVP is rare. A single IV dose of desmopressin adjusted to the patient's weight is safe as pre-PRB bleeding prophylaxis.

Comparison of in situ preservation techniques for kidneys from donors after circulatory death: a systematic review and meta-analysis.

BACKGROUND: Several techniques have been developed to reduce the warm ischaemic injury of donation after circulatory death (DCD) organs before procurement. There are scarce data about the in situ preservation techniques for kidney graft outcomes. The aim of this systematic review was to evaluate the best in situ preservation method for kidney graft outcomes from organs obtained from controlled and uncontrolled DCD. METHODS: A systematic review of the PubMed (MEDLINE), Embase, LILACS and Cochrane databases was conducted. Studies that compare two or more in situ preservation techniques were identified and included. Only studies which provided enough data to calculate odds ratio were eligible for meta-analysis. PROSPERO registration: CRD42020179598. RESULTS: The search strategy yielded 7,121 studies. Ultimately, 14 retrospective studies were included. Because of heterogeneity, the included studies provided weak evidence that normothermic regional perfusion (NRP) is the best in situ preservation technique in terms of delayed graft function (DGF) rates. Regarding primary nonfunction (PNF), we carried out a meta-analysis of 10 studies with a pooled OR of 0.83 (95% CI: 0.40-1.71), for the NRP. In regard to DGF, pooled OR for NRP was 0.36 (95% CI: 0.25-0.54). CONCLUSIONS: NRP in the DCD donor could improve kidney graft function and be considered the in situ preservation technique of choice for abdominal organs.

Absolute blood volume variations and vascular refilling in hemodialysis patients.

The imbalance between ultrafiltration volume (UF) and vascular refilling is considered a major cause for intradialytic hypotension. Recent studies report a noninvasive method to estimate vascular refilling (VREF ) by determining absolute blood volume (ABV). It was the aim of the study to analyze variations in ABV in a group of hemodialysis (HD) patients and examine VREF . Thirty one stable chronic HD patients were studied, aged 71.07 ± 13.31 years. Dialysis duration and UF requirements were based on physician prescription. VREF was calculated as: VREF  = VUF  - ΔV where ΔV is ABV variation during dialysis treatment. ABV at the beginning of the dialysis was 6.00 ± 2.39 L (92.82 ± 33.17 ml/kg) and at the end 5.38 ± 2.32 L (82.07 ± 31.41 ml/kg). Prescribed UF was 2.64 ± 0.83 L. Mean VREF was 2.05 ± 0.80 L, with a refilling fraction of 75.75 ± 12.79%. VREF was strongly correlated with UF volume (r2 0.877), and with pre-dialysis volume overload (r2 0.617). Patients under beta-blocker treatment showed significantly lower FREF . ABV measurement is an easy and noninvasive method that allows us to study VREF during HD. We found a strong correlation between VREF and UF.

Absolute blood volume variations during hemodialysis: Does dialysate temperature play a role?

BACKGROUND: Vascular refilling occurs to preserve hemodynamic stability during hemodialysis (HD). Recent studies report a feasible and noninvasive method to determine absolute blood volume (ABV), and estimate vascular refilling during HD. The objective of this study is to analyze if lowering dialysate temperature modifies variations in ABV during HD. METHODS: The study was performed in 50 patients under HD. During two different sessions, relative blood volume was assessed using dialysate temperatures of 35.5°C (cool dialysate) and 36.5°C (neutral dialysate). ABV and vascular refilling were calculated using Kron et al methodology. RESULTS: Thirty-nine intradialytic morbid events (IMEs) were observed in 30 patients, 14 under cool dialysate and 25 during neutral dialysate. We did not found statistically differences in ABV or in refilling volume between cool and neutral temperature. When analyzing apart only those patients who presented IME, we observed lower drop in ABV in the 35.5°C dialysate treatments (0.57 L) versus 36.5°C dialysate treatments (0.71 L). When cool dialysate was used, the vascular refilling fraction tended to be higher, but data did not turn statistically significant. CONCLUSIONS: In selected groups of patients the use of cool dialysate induces lower ABV variations that could improve hemodynamic stability during HD treatments.

Relapsing peritonitis and taurolidine peritoneal catheter lock: One center experience.

BACKGROUND: Relapsing peritonitis due to the development of a biofilm in the catheter's lumen remains an important complication of peritoneal dialysis therapy that endangers technique continuity. Taurolidine catheter lock has proven efficient reducing infection rates in permanent hemodialysis catheters based on its biocidal activity and biofilm detachment effect. Efficacy evidence on its use in peritoneal dialysis catheters is lacking. METHODS: We retrospectively analyzed all relapsing peritonitis episodes from June 2018 until October 2019 in our center. Patients were identified and data were collected from our electronic renal registry and patient's records. RESULTS: Six patients were identified during the study period. Most patients (66.6%) were on automated peritoneal dialysis and the median duration of peritoneal dialysis before the episode of taurolidine was started was 43.66 ± 29.64 months. Mean taurolidine doses were 10 (range: 9-11) and 83.3% (five patients, with peritonitis caused by Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Corynebacterium propinquum) had a favorable response and microbial eradication without relapses after taurolidine treatment. Only one patient relapsed by the same organism (Corynebacterium amycolatum) due to non-adherence to the antibiotic treatment prescribed. None of the patients experienced any relevant adverse events, with only two out of six presenting mild transient abdominal discomfort. CONCLUSION: We believe that peritoneal catheter taurolidine lock could be considered in cases of relapsing or refractory peritonitis, as it could prevent catheter removal and permanent switch to hemodialysis in selected cases, although literature is scarce and further studies are needed.

Taurolidine Peritoneal Dialysis Catheter Lock to Treat Relapsing Peritoneal Dialysis Peritonitis.

Peritonitis remains a primary challenge for the long-term success of peritoneal dialysis (PD) technique and one of the main reasons for catheter removal. Prevention and treatment of catheter-related infections are major concerns to avoid peritonitis. The use of taurolidine catheter-locking solution to avoid the development of a biofilm in the catheter's lumen has obtained good results in hemodialysis catheters for reducing infection rates, although there is scarce literature available regarding its utility in PD. We describe the case of a woman in her 60s who developed relapsing peritonitis due to Pseudomonas aeruginosa, with no possibility of removing peritoneal dialysis catheter because she was not a suitable candidate for hemodialysis. After the fourth peritonitis episode caused by Pseudomonas species, the use of taurolidine catheter-locking solution was initiated. She received a total of 9 doses, with a favorable microbiological and clinical outcome and no further relapses more than 10 months after taurolidine PD catheter lock treatment was started. We report the successful elimination of an aggressive bacteria after taurolidine PD catheter lock use, with no relevant adverse events.

Structure-Driven Discovery of α,γ-Diketoacid Inhibitors Against UL89 Herpesvirus Terminase.

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing a variety of severe viral infections, including irreversible congenital disabilities. Nowadays, HCMV infection is treated by inhibiting the viral DNA polymerase. However, DNA polymerase inhibitors have several drawbacks. An alternative strategy is to use compounds against the packaging machinery or terminase complex, which is essential for viral replication. Our discovery that raltegravir (1), a human immunodeficiency virus drug, inhibits the nuclease function of UL89, one of the protein subunits of the complex, prompted us to further develop terminase inhibitors. On the basis of the structure of 1, a library of diketoacid (α,γ-DKA and ß,δ-DKA) derivatives were synthesized and tested for UL89-C nuclease activity. The mode of action of α,γ-DKA derivatives on the UL89 active site was elucidated by using X-ray crystallography, molecular docking, and in vitro experiments. Our studies identified α,γ-DKA derivative 14 able to inhibit UL89 in vitro in the low micromolar range, making 14 an optimal candidate for further development and virus-infected cell assay.

Structure of the homodimeric androgen receptor ligand-binding domain.

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.

Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain.

During viral replication, herpesviruses package their DNA into the procapsid by means of the terminase protein complex. In human cytomegalovirus (herpesvirus 5), the terminase is composed of subunits UL89 and UL56. UL89 cleaves the long DNA concatemers into unit-length genomes of appropriate length for encapsidation. We used ESPRIT, a high-throughput screening method, to identify a soluble purifiable fragment of UL89 from a library of 18,432 randomly truncated ul89 DNA constructs. The purified protein was crystallized and its three-dimensional structure was solved. This protein corresponds to the key nuclease domain of the terminase and shows an RNase H/integrase-like fold. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn(2+) ions, two of which are located at the active site pocket. We also show that the nuclease function can be inactivated by raltegravir, a recently approved anti-AIDS drug that targets the HIV integrase.