Design and Optimization of an α-Helical Bundle Dimer Cell-Penetrating Peptide for In Vivo Drug Delivery.

To deliver membrane-impermeable drugs into eukaryotic cells, a lot of cell-penetrating peptides (CPPs) were discovered. Previously we designed an amphipathic α-helical peptide which dimerizes itself via its two C-residues. This bis-disulfide-linked dimeric bundle, LK-3, has remarkable cell-penetrating ability at nanomolar concentration, which is an essential prerequisite for CPP. In an effort to optimize the sequence of LK-3, we adjusted its length and evaluated changes in the dimerization rate. We found that a 10-amino-acid monomer has the fastest dimerization rate and subsequently modified its hydrophobic and hydrophilic residues to construct a small peptide library. The evaluation of cell permeability of these derivatives showed that their cell-penetrating ability is comparable to that of the LK-3, except V- or H-containing ones. In this library, diLR10 was found to display fast nanomolar cell membrane penetration, low toxicity, and ease of production. The methotrexate (MTX) conjugate of diLR10, MTX-diLR10, has a 19-fold increased efficacy over MTX in MDA-MB-231 cells and efficiently deflates lesions in a rheumatoid arthritis (RA) in vivo mouse model.

Modified retroperitoneal lymph node dissection for post-chemotherapy residual tumour: a long-term update.

OBJECTIVE: To update previously reported outcomes of modified-template post-chemotherapy retroperitoneal lymph node dissection (PC-RPLND) in appropriately selected patients with metastatic non-seminomatous germ cell tumour (NSGCT), as our previous report was criticised for short follow-up and so we now provide a long-term update on this cohort. PATIENTS AND METHODS: In all, 100 patients with normal serum markers after cisplatin-based chemotherapy and residual retroperitoneal tumour underwent modified PC-RPLND between 1991 and 2004. Using a prospectively managed institutional testicular cancer database, long-term follow-up was obtained. RESULTS: As previously reported, 43 patients underwent a right-modified template, 18 patients underwent a full-left-modified template, and 39 patients underwent a left-modified template. The updated long-term median follow-up for the entire cohort is 125 months. Seven patients developed recurrent disease with a median (range) time to recurrence of 11 (6-102) months, and one patient died from recurrent disease in the chest 4 years after surgery. All recurrences were outside the boundaries of a full-bilateral template RPLND, with the most common location of recurrence being the chest. The 5- and 10-year recurrence-free survival rates were 93% and 92%, respectively. The overall survival at 10 years was 99%. CONCLUSIONS: In appropriately selected patients with low-volume disease before and after chemotherapy, a modified template has durable long-term efficacy without risk of in-field recurrences at a median follow-up of 125 months.

Technique for office-based, ultrasonography-guided percutaneous biopsy of renal cortical neoplasms using a novel transducer for facilitated ultrasound targeting.

OBJECTIVES: To help clarify which small renal cortical neoplasms (RCNs) require surgery by using office-based, ultrasonography-guided percutaneous renal biopsy. PATIENTS AND METHODS: Biopsies were performed using facilitated ultrasound targeting (FUT) technology, which incorporates a needle guide and onscreen beam-steered technology to permit highly precise needle deployment. Patient and tumour characteristics, procedure time, complications and biopsy efficacy were documented. Wong-Baker pain levels were obtained before, during and 1 h after the procedure. RESULTS: Seven patients underwent biopsy, six for RCNs and one for medical renal disease. The mean (range) patient age was 68.5 (54-79) years, and the mean (range) tumour diameter was 2.55 (2.0-2.9) cm. Mean pain levels before, during and 1 h after the procedure were 0, 1.6 and 0.5, respectively. There were no intra- or post-procedural complications. Biopsy results were diagnostic in five of the six RCN cases and in the single case of medical renal disease. CONCLUSIONS: Our preliminary experience shows that office-based percutaneous renal biopsy using a novel transducer for FUT is safe and effective. An international multicentre study is planned to confirm these preliminary results.

Neoadjuvant chemotherapy in urothelial bladder cancer: impact of regimen and variant histology.

AIM: We compared the efficacy of methotrexate/vinblastine/doxorubicin/cisplatin (MVAC) versus gemcitabine/cisplatin in urothelial cancer and neoadjuvant chemotherapy (NACT) efficacy in variant histology (VH). MATERIALS & METHODS: Radical cystectomy patients were retrospectively compared with those who received NACT. Factors associated with survival, pathologic complete response (pCR) and downstaging (pDS) were evaluated in multivariable models. RESULTS: 9% of radical cystectomy patients (84/919) received NACT, with improved survival, pCR and pDS on both regimens. MVAC lead to higher pDS without an increase in pCR. On multivariable analysis, there was a nonsignificant increase in pDS with MVAC. NACT conferred similar responses in squamous and glandular differentiation VH. CONCLUSION: NACT was associated with improved survival, pCR and pDS. Furthermore, responses to NACT were not dependent on presence of VH.

Consideration of comorbidity in risk stratification prior to prostate biopsy.

BACKGROUND: Previously, the patient-reported Total Illness Burden Index for Prostate Cancer (TIBI-CaP) questionnaire and/or the physician-reported Charlson Comorbidity Index (CCI) have provided assessments of competing comorbidity during treatment decisions for patients with prostate cancer. In the current study, the authors used these assessments to determine comorbidity and prognosis before prostate biopsy and the subsequent diagnosis of prostate cancer to identify those patients least likely to benefit from treatment. METHODS: A prospective observational cohort study was performed of 104 participants aged 64.0 years ± 6.5 years from 3 institutions representing different health care delivery systems. Patients were identified before undergoing transrectal ultrasound-guided prostate biopsy and followed for a median of 28 months. Associations between the comorbidity scores and nonelective hospital admissions were investigated using logistic regression and Cox proportional hazards models. RESULTS: Among the 104 patients who underwent prostate biopsy, 2 died during the follow-up period. The overall hospital admission rate was 20% (21 of 104 patients). Higher scores on both the TIBI-CaP (≥ 9) and CCI (≥ 3) were found to be significantly associated with an increased odds for hospital admission (odds ratio, 11.3 [95% confidence interval (95% CI), 2.4-53.6] and OR, 5.7 [95% CI, 1.4-22.4]) and hazards ratios (HRs) for time to hospital admission (HR, 3.8 [95% CI, 1.3-11.2] and HR, 3.2 [95% CI, 1.1-9.1]), respectively. CONCLUSIONS: TIBI-CaP and CCI scores were found to successfully predict which patients were at high risk for nonelective hospital admission. These patients are likely to have poorer health and a potentially shortened lifespan. Therefore, comorbidity analysis using these tools may help to identify those patients who are least likely to benefit from prostate cancer therapy and should avoid prostate biopsy.

Severity of hematuria effects resolution in patients treated with hyperbaric oxygen therapy for radiation-induced hematuria.

INTRODUCTION: We investigated the differences between prostate cancer patients with radiation-induced hematuria treated with hyperbaric oxygen (HBO) therapy that did or did not have a resolution of hematuria. MATERIALS AND METHODS: We performed a retrospective review of prostate cancer patients with radiation-induced hematuria who underwent HBO from April 2000 to March 2010. We performed an analysis of demographic data and severity of hematuria in those who had resolution of or persistent hematuria. Additionally, prostate-specific antigen (PSA) data were also obtained during the study period. RESULTS: Overall, 11/22 men had resolution of hematuria after HBO therapy with a median follow-up of 2.2 (0.35-13.6) years. The Radiation Therapy Oncology Group (RTOG) grade of hematuria is predictive of final hematuria outcome (resolution vs. persistent) after HBO (p = 0.026). No significant PSA changes were noted before and after HBO therapy. CONCLUSIONS: The RTOG hematuria grade is associated with the resolution of hematuria after HBO therapy for radiation-induced hematuria in men treated for prostate cancer. This information may be helpful during shared medical decision-making regarding utility of HBO therapy in the context of severity of hematuria.

Comparative analysis of open government data topics and usability.

This study identifies differences in the content of open public data managed by the central government, local governments, public institutions, and the office of education in Korea through keyword network analysis. Pathfinder network analysis was performed by extracting keywords assigned to 1,200 data cases, open to the Korean Public Data Portals. Subject clusters were derived for each type of government and their utility was compared using download statistics. Eleven clusters were formed for public institutions with specialized information on national issues such as Health care and Real estate, while 15 clusters were formed for the central government with national administrative information, including Crime and Safety policing. Local governments and offices of education were assigned 16 and 11 topic clusters respectively, with data focusing on regional life such as Local factories and manufacturing, Resident registration, and Lifelong education. Usability was higher in public and central governments that deal with national-level specialized information than for regional-level information. It was also confirmed that subject clusters such as Health care, Real estate, and Crime showed high usability. Furthermore, there was a large gap in data utilization because of the existence of popular data that showed extremely high usage. Supplementary Information: The online version contains supplementary material available at 10.1007/s11135-023-01630-x.

Managing Human Subjects Research During a Global Pandemic at an Academic Center: Lessons Learned From COVID-19.

By March 2020, New York City became the early epicenter of the COVID-19 pandemic in the United States. Consequently, Columbia University, with its large portfolio of human subjects research, had to address the challenges of protecting thousands of research participants and research staff from potential exposure to COVID-19 while facilitating essential biomedical research, especially pandemic-related studies. The authors describe, from the perspective of Columbia's research administration leadership, how the University and its primary teaching hospital ramped down-and later ramped up-human subjects research and reflect on lessons learned. As the pandemic unfolded, only studies offering the prospect of direct benefit to subjects were permitted to continue with in-person contact. New in-person participant enrollment ceased, except for COVID-19 prevention or treatment studies. Centralized, frequently updated communication about policies and procedures was disseminated to the Columbia research community. Procedural efficiencies were rapidly developed and implemented for review and oversight of human subjects research and contractual agreements for clinical trials. More frequent institutional review board meetings and 24-hour support markedly reduced turnaround time for COVID-19 studies, without delaying approvals of non-COVID-19 research. Research administration worked closely with relevant principal federal agencies, whose regulatory flexibility facilitated the efficient implementation of COVID-19-related research. Overall, the ramp-down and ramp-up of the majority of human subjects research, with specified priorities and accelerated processes, worked well. Adjustments were made to handle the increase in administrative tasks, the need to respond rapidly to added oversight responsibilities, and the management of the many new COVID-19-related research protocols. Timely, centralized communication, support for staff needs, prioritization, and collaboration were critical to successful research oversight at a large-scale academic setting in the midst of a pandemic. These perspectives may be useful to academic research centers addressing the current and future pandemics.

Surgical site infection rates following laparoscopic urological procedures.

PURPOSE: Surgical site infections have been categorized by the Centers for Medicare and Medicaid Services as "never events". The incidence of surgical site infection following laparoscopic urological surgery and its risk factors are poorly defined. We evaluated surgical site infection following urological laparoscopic surgery and identified possible factors that may influence occurrence. MATERIALS AND METHODS: Patients who underwent transperitoneal laparoscopic procedures during a 4-year period by a single laparoscopic surgeon were retrospectively reviewed. Surgical site infections were identified postoperatively and defined using the Centers for Disease Control criteria. Clinical parameters, comorbidities, smoking history, preoperative urinalysis and culture results as well as operative data were analyzed. Nonparametric testing using the Mann-Whitney U test, multivariable logistic regression and Spearman's rank correlation coefficient were used for data analysis. RESULTS: In 556 patients undergoing urological laparoscopic procedures 14 surgical site infections (2.5%) were identified at mean postoperative day 21.5. Of the 14 surgical site infections 10 (71.4%) were located at a specimen extraction site. Operative time, procedure type and increasing body mass index were significantly associated with the occurrence of surgical site infections (p = 0.007, p = 0.019, p = 0.038, respectively), whereas history of diabetes mellitus (p = 0.071) and intraoperative transfusion (p = 0.053) were found to trend toward significance. Age, gender, positive urine culture, steroid use, procedure type and smoking history were not significantly associated with surgical site infection. Body mass index and operative time remained significant predictors of surgical site infection on multivariate logistic regression analysis. CONCLUSIONS: Surgical site infection is an infrequent complication following laparoscopic surgery with the majority occurring at the specimen extraction site. Infection is associated with prolonged operative time and increasing body mass index.

Ocular Ultrasound Abnormalities and Optic Nerve Sheath Diameter in Dogs and Cats.

Indications for, technique, and findings for normal and abnormal ocular ultrasound are discussed, with specific sonographic findings, images, differential diagnoses, and other considerations. Because the eye is a fluid-filled structure, ultrasound can be used as a screening test when pathology prevents direct examination. Structural abnormalities, such as lens luxation, retinal detachments, and intraocular and orbital masses, also may be defined better using point-of-care ultrasound. Details on additional ophthalmic diagnostics, treatment, and prognosis are not covered.

RAPIDLY PROGRESSIVE NEOVASCULAR GLAUCOMA FROM CYTOMEGALOVIRUS RETINITIS IN A NON-HUMAN IMMUNODEFICIENCY VIRUS PATIENT.

PURPOSE: To describe a case of neovascular glaucoma from cytomegalovirus (CMV) retinitis in a human immunodeficiency virus-negative patient with immunosuppression after stem-cell transplant for multiple myeloma. METHODS: Retrospective case report. RESULTS: A 71-year-old man on monthly infusion of daratumumab for multiple myeloma after stem-cell transplant presenting with a 2-week history of floaters, photophobia, and blurry vision was found to have polymerase chain reaction-confirmed CMV retinitis associated with diffuse occlusive vasculitis. The patient was human immunodeficiency virus negative with a CD4 count of 450/mm3. Despite immediate aggressive treatment, the patient developed neovascular glaucoma with poor visual outcome. CONCLUSION: Cytomegalovirus retinitis in human immunodeficiency virus-negative patients is becoming more prevalent with increasing use of systemic immunosuppression therapy for various reasons. Patients with non-human immunodeficiency virus related CMV retinitis can have severe ischemia atypical of the classic CMV retinitis and should be followed closely for neovascularization.

Cytokine Levels in Human Vitreous in Proliferative Diabetic Retinopathy.

In this study, we compare the vitreous cytokine profile in patients with proliferative diabetic retinopathy (PDR) to that of patients without PDR. The identification of novel cytokines involved in the pathogenesis of PDR provides candidate therapeutic targets that may stand alone or work synergistically with current therapies in the management of diabetic retinopathy. Undiluted vitreous humor specimens were collected from 74 patients undergoing vitrectomy for various vitreoretinal disorders. Quantitative immunoassay was performed for a panel of 36 neuroinflammatory cytokines in each specimen and assessed to identify differences between PDR (n = 35) and non-PDR (n = 39) patients. Levels of interleukin-8 (IL-8), IL-15, IL-16, vascular endothelial growth factor (VEGF), VEGF-D, c-reactive protein (CRP), serum amyloid-A (SAA), and intracellular adhesion molecule-1 (ICAM1) were significantly increased in the vitreous of PDR patients compared to non-PDR patients (p < 0.05). We report novel increases in IL-15 and IL-16, in addition to the expected VEGF, in the human vitreous humor of patients with PDR. Additionally, we confirm the elevation of ICAM-1, VCAM-1, SAA, IL-8 and CRP in the vitreous of patients with PDR, which has previously been described.

Followup imaging after urological imaging studies: comparison of radiologist recommendation and urologist practice.

PURPOSE: We determined if differences in the perceived need for followup imaging between an ordering urologist and the interpreting radiologist have an impact on the use of imaging technology for urological conditions. MATERIALS AND METHODS: Consecutive radiology reports for 985 patients were retrospectively reviewed in 2 urological practices. Imaging included computerized tomography, magnetic resonance imaging, ultrasonography and excretory urography. All imaging reports were analyzed for the radiologist recommendation for followup imaging and correlated with subsequent studies ordered by the urologist within 6 months of the original study. RESULTS: Radiologists recommended followup studies in 202 of 985 reports (20.5%). A followup study was actually ordered for 65 of these 202 patients (32.2%). Urologists ordered studies for 87 of 783 (11.1%) patients for whom the radiologist did not make a recommendation. Overall urologists ordered followup studies for 152 of 985 patients (15.4%) or 24.8% fewer studies than recommended by the radiologist. CONCLUSIONS: There is a significant reduction in imaging use when urologists evaluate radiologist recommendations and then direct followup imaging.

Patient noncompliance before surgery.

OBJECTIVE: To assess patient compliance with preoperative instructions and to determine causes and factors of noncompliance, as noncompliance might affect surgical outcome and has potential medicolegal implications. PATIENTS AND METHODS: One surgeon counselled 101 consecutive patients before undergoing laparoscopic renal surgery. Deliberate discussions instructed patients to bring their radiograph films on the day of surgery and complete a preoperative bowel preparation. Noncompliance was defined as failure to bring films and/or complete bowel preparation. Patient demographics, socio-economic and clinical variables were analysed, and reasons for failure to comply with instructions were also recorded. RESULTS: Twenty-four of the 101 (24%) patients were not compliant, 13 with films only, seven with bowel preparation only, and four with both sets of instructions. Univariate analysis showed that language and race were factors for noncompliance. Multivariate analysis showed that non-Caucasians had 17 times the risk of noncompliance (P < 0.001); long distance from home to the site of care had five times the risk of noncompliance (P = 0.041), and each day between the initial consultation and the date of surgery had 1.05 times the risk of noncompliance (P < 0.001). The most common reason given by patients for noncompliance was that they were never given the preoperative instructions. CONCLUSIONS: Noncompliance with preoperative surgical instructions is a significant issue and is increased in non-Caucasian patients, those travelling long distances, and those whose surgery date is long after their preoperative consultation. A systems-based approach is needed to address this significant issue.

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

Influence of hypoxia on the stemness of umbilical cord matrix-derived mesenchymal stem cells cultured on chitosan films.

Chitosan is attractive as a substrate for stem cell expansion because it improves stemness through formation of spheroids. Hypoxia has also been proposed as a strategy to enhance stemness and survival of stem cells after in vivo implantation. This study was therefore designed to evaluate the influence of hypoxia on chitosan-induced behavior of stem cells. Umbilical cord matrix-derived stem cells were cultured on chitosan film or standard plate under normoxia and hypoxia, for 3 and 7 days. Based on immunophenotyping, chitosan strongly suppresses the expression of CD90 and CD105 cell surface markers, changes partially reversed by combined exposure to hypoxia. Hypoxia generally increased the volume and number of spheroids formed on chitosan, but the cellularity of cultures on chitosan films remained lower than that of standard plates. After 7 days of culture, the expression of stemness related genes (Oct4, Sox2, and Nanog) was best stimulated by combined exposure to chitosan and hypoxia. Based on our results, conditioning stem cells for 7 days on chitosan films under hypoxic conditions is recommended to enhance the stemness of stem cells, and minimize cell loss due to lack of attachment on chitosan. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 501-511, 2018.

Oligomer Formation Propensities of Dimeric Bundle Peptides Correlate with Cell Penetration Abilities.

LK-3, an amphipathic dimeric peptide linked by two disulfide bonds, and related isomeric bundles were synthesized, and their cell penetrating abilities were investigated. The measurements using size exclusion chromatography and dynamic light scattering show that LK-3 and its isomers form cell penetrating oligomers. Calculations, performed for various types of peptide isomers, elucidate a strong correlation between the amphipathic character of dimers and cell penetration ability. The results suggest that the amphipathicities of LK-3 and related bundle dimers are responsible for their oligomerization propensities which in turn determine their cell penetrating abilities. The observations made in this study provide detailed information about the mechanism of cell uptake of LK-3 and suggest a plausible insight of the early stage of nanoparticle formation of the cell penetrating amphipathic peptides.

Use of the cell cycle progression (CCP) score for predicting systemic disease and response to radiation of biochemical recurrence.

BACKGROUND: Determining the optimal treatment for biochemical recurrence (BCR) after radical prostatectomy (RP) is challenging. OBJECTIVE: We evaluated the ability of CCP score (a prognostic RNA expression signature) to discriminate between systemic disease and local recurrence in patients with BCR after RP. METHODS: Sixty patients with BCR after RP were selected for analysis based on: 1) metastatic disease, 2) non-response to salvage external beam radiotherapy (EBRT), and 3) durable response to salvage EBRT. CCP scores were generated from the RNA expression of 46 genes. Logistic regression assessed the association between CCP score and patient group. RESULTS: Passing CCP scores were generated for 47 patients with complete clinical and pathologic data. CCP score predicted clinical status when comparing patients with metastatic disease or non-responders to salvage therapy to patients with durable response (p = 0.006). CCP score remained significantly predictive of clinical status after accounting for time to BCR, PSA level at BCR, and Gleason score (p = 0.0031). CONCLUSIONS: Elevated CCP score was associated with increased risk of systemic disease, indicating that CCP score may be useful in identifying patients with BCR who are most likely to benefit from salvage radiation therapy.

Rapid generation of sub-type, region-specific neurons and neural networks from human pluripotent stem cell-derived neurospheres.

Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation, but they often clump in culture, which has always represented a challenge for neurodifferentiation. In this study, we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM) with 10% CO2, which doubled the expression of the NESTIN, PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore, an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the "neurosphederm". The large neural tube-type rosette (NTTR) structure formed from the neurosphederm, and the NTTR expressed higher levels of the PAX6, SOX2 and NESTIN genes compared with the neuroectoderm-derived neuroprogenitors. Different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic neurons appeared within 27 days using the neurosphederm, which is a shorter period than in traditional neurodifferentiation-protocols (42-60 days). With additional supplements and timeline dopaminergic and Purkinje neurons were also generated in culture too. Furthermore, our in vivo results indicated that the fragmented neurospheres facilitated significantly better neurogenesis in severe combined immunodeficiency (SCID) mouse brains compared with the non-fragmented neurospheres. Therefore, this neurosphere-based neurodifferentiation protocol is a valuable tool for studies of neurodifferentiation, neuronal transplantation and high throughput screening assays.