Formulation and Skin Permeation of Active-Loaded Lipid Nanoparticles: Evaluation and Screening by Synergizing Molecular Dynamics Simulations and Experiments.

Encapsulation into nanoparticles (NPs) is a potential method to deliver pharmaceutical/cosmetic actives deep into the skin. However, understanding the NP formulations and underlying mechanism of active delivery to skin has scarcely been studied. We report a simulation platform that screens, evaluates, formulates, and provides atomic-resolution interpretation of NP-based formulations, and reveals the active permeation mechanism from NPs to skin. First, three actives, namely, ferulic acid (FA), clotrimazole (CZE), and tretinoin (TTN), and five lipid excipients' (Compritol, Precirol, Geleol, Gelot, Gelucire) combinations were screened by MD simulations for the best pairs. For each suggested pair, the actual active and lipid compositions for the synthesis of stable NP formulations were then obtained by experiments. MD simulations demonstrate that in NP formulations, FA and CZE actives are present at the surface of the NPs, whereas TTN actives are present at both the surface and interior of the NP core. The NP shapes obtained by simulation perfectly match with experiments. For each NP, separate MD simulations illustrate that active-loaded NPs approach the skin surface quickly, and then actives translocate from NP surface to skin surface followed by penetration of NPs through skin. The driving force for the translocation which initiates during the penetration process, is the stronger active-skin interaction compared to active-NP interaction. Permeation free energy indicates spontaneous transfer of actives from solution phase to the surface of the skin bilayer. The free energy barriers are increased in the order of FA < TTN < CZE. Significantly lower diffusions of actives are obtained in the main barrier region compared to bulk, and the average diffusion coefficients of actives are in the same order of magnitude (∼10-6 cm2/s). The estimated permeability coefficients (log P) of actives are mainly governed by free energy barriers. The study would facilitate the development of novel lipid-based NP formulations for personal-care/pharmaceutical applications.

Balancing the Hype with Reality: What Do Patients with Advanced Melanoma Consider When Making the Decision to Have Immunotherapy?

BACKGROUND: Immunotherapy has resulted in unprecedented improvements in survival and maintained quality of life for many patients with advanced melanoma. However, durable responses are observed in only a minority of patients, and severe treatment side effects are experienced by 5%-30%. There are no reliable tests that can differentiate between patients who are likely to respond to immunotherapy and those who will not. Hence, new challenges have arisen as clinicians try to facilitate patients in their decision-making regarding immunotherapy. Furthermore, little is known about the real-world patients' experience and understanding of immunotherapy outside the clinical trial setting. Here, we explore the perspectives of patients undergoing immunotherapy for melanoma and focus on factors that influenced their treatment decision-making. MATERIALS AND METHODS: Twenty-three in-depth semistructured interviews were conducted with patients receiving pembrolizumab for stage IV melanoma at an Australian public cancer hospital. Patients were recruited at a range of time points after commencing therapy, and their experience of treatment was explored. Interviews were audio recorded, transcribed verbatim, coded, and analyzed thematically. RESULTS: Immunotherapy is viewed as a symbol of hope, with high-profile anecdotes reinforcing this perception. Only a minority of patients expressed a good understanding of the likely efficacy and potential treatment side effects. Patients are reliant on their clinicians' recommendation regarding immunotherapy treatment decisions. CONCLUSION: Novel treatments such as immunotherapy provide significant hope for patients. This may influence their preference for immunotherapy over and above the usual considerations of the trade-off between efficacy and toxicity. Careful counsel and individualized patient resources may further facilitate treatment decision-making. IMPLICATIONS FOR PRACTICE: This study highlighted some of the misconceptions held by patients that need to be addressed when discussing the possibility of receiving treatment with immunotherapy for advanced melanoma. Patients placed a lot of importance on high-profile anecdotes rather than truly understanding likely outcomes of treatment based on personal circumstances. The majority of patients had a poor understanding of the potential side effects and long-term implications of treatment with immunotherapy. Careful counsel is required in order to facilitate informed decision-making about treatment and to ensure possible side effects are known and appreciated. Further research is needed to develop tools to aid decision-making in everyday clinical practice.

Efficacy of anti-PD-1 therapy in patients with melanoma brain metastases.

BACKGROUND: There is limited data on the efficacy of anti-programmed death 1 (PD-1) antibodies in patients (pts) with melanoma brain metastasis (BM), particularly those which are symptomatic. METHOD: We retrospectively assessed pts with melanoma BM treated with PD-1 antibodies, nivolumab and pembrolizumab. Clinicopathologic and treatment parameters were collected and outcomes determined for intracranial (IC) response rate (RR) using a modified RECIST criteria, with up to five IC target lesions used to determine IC response, disease control rate (DCR) and progression-free survival (PFS). RESULTS: A total of 66 pts were identified with a median follow up of 7.0 months (range 0.8-24.5 months). A total of 68% were male and 45% BRAF V600 mutation positive. At PD-1 antibody commencement, 50% had an elevated LDH; 64% had local therapy to BM prior to commencing anti-PD1, of which 5% had surgical resection, 14% stereotactic radiosurgery (SRS), 18% whole-brain radiotherapy (WBRT), 27% had surgery and radiotherapy. Twenty-one per cent started anti-PD-1 as first line systemic therapy. No pt had prior anti-PD-1 treatment. The IC overall RR was 21 and DCR 56%. Responses occurred in 21% of pts with symptomatic BM. The median OS was 9.9 months (95% CI 6.93-17.74). Pts with symptomatic BM had shorter PFS than those without symptoms (2.7 vs 7.4 months, P=0.035) and numerically shorter OS (5.7 vs 13.0 months, P=0.068). Pts requiring corticosteroids also had a numerically shorter PFS (3.2 vs 7.4 months, P=0.081) and OS (4.8 vs 13.1 months, P=0.039). CONCLUSIONS: IC responses to anti-PD-1 antibodies occur in pts with BM, including those with symptomatic BM requiring corticosteroids. Prospective trials evaluating anti-PD-1 therapy in pts with BM are underway.

The Advantages and Challenges of Using FDG PET/CT for Response Assessment in Melanoma in the Era of Targeted Agents and Immunotherapy.

The treatment of melanoma has been revolutionised in recent years by advances in the understanding of the genomic landscape of this disease, which has led to the development of new targeted therapeutic agents, and the ability to therapeutically manipulate the immune system through inhibition of cancer cell-T-cell interactions that prevent an adaptive immune response. While these therapeutic interventions have dramatically improved the prospects of survival for patients with advanced melanoma, they bring significant complexity to the interpretation of therapeutic response because their mechanisms and temporal profile of response vary considerably. In this review, we discuss the mode of action of these emerging therapies and their toxicities to provide a framework for the use of FDG PET/CT in therapeutic response assessment. We propose that the greatest utility of PET in assessment of response to agents that abrogate signalling related to BRAF mutation is for early assessment of resistance, while in anti-CTLA4 therapy, immunological flare can compromise early assessment of response but can identify potentially life-threatening autoimmune reactions. For anti-PD1/PDL1 therapy, the role of FDG PET/CT is more akin to its use in other solid malignancies undergoing treatment with conventional chemotherapy. However, further research is required to optimise the timing of scans and response criteria in this disease.

Evidence supporting the 19 ß-strand model for Tom40 from cysteine scanning and protease site accessibility studies.

Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a ß-barrel containing 19 membrane spanning ß-strands and an N-terminal α-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential ß-strands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 ß-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space.

Ambient light spectrum affects larval Mexican jumping bean moth (Cydia saltitans) behavior despite light obstruction from host seed.

Spectral differences in ambient light can affect animal behavior and convey crucial information about an individual's environment. The ability to perceive and respond to differences in ambient light varies widely by taxa and is shaped by a species' ecology. Mexican jumping bean moths, Cydia saltitans, spend their entire larval period encased in fallen host seeds and contend with potentially lethal environmental temperatures when host seeds are in direct sunlight. We investigate if and how C. saltitans larvae in host seeds respond to lighting conditions associated with these thermal risks. In a temperature-controlled experiment, we identified that larvae demonstrated distinct behavioral ("jumping") responses corresponding to four lighting treatments (white, red, green, and purple), despite extremely minimal light penetration through host seed walls. Red light induced the greatest larval activity (measured by probability of movement and by displacement from origin), suggesting that larvae have mechanisms to perceive low levels of red light and/or to detect subtle increases in heat produced by red/near infrared-biased light spectra, possibly providing them with an early-warning mechanism against thermal stress. Our findings highlight the interplay of environmental lighting, behavior, and potential thermosensory adaptations in a species with a visually constrained environment.

Real-World Outcomes of Immunotherapy for Melanoma Brain Metastases in New Zealand.

PURPOSE: Melanoma brain metastases (BMs) are associated with poor survival. Combination immune checkpoint inhibitors (ICIs) with anti-PD1 and anti-CTLA-4 are the international standard-of-care treatment. Most landmark clinical trials excluded real-world patients with symptomatic disease, poor performance status (PS), and steroid use. Despite the high incidence of melanoma in New Zealand (NZ), the only publicly funded systemic treatment is anti-PD1 monotherapy. The real-world outcomes for BMs after ICIs in NZ are unknown. METHODOLOGY: Medical records of patients with melanoma BMs in seven cancer centers across NZ between September 1, 2016, and September 1, 2020, were evaluated. Clinicopathologic characteristics, treatment, intracranial (IC) tumor response rates, IC progression-free survival, and overall survival (OS) are reported. RESULTS: One hundred and forty-four patients received at least one dose of ICI. One hundred and thirty-three (93%) patients received anti-PD1 monotherapy. Almost a quarter of patients had poor baseline PS, 56% were symptomatic, and 33% had corticosteroids. Patients also received local therapies: 61 (42%) patients underwent surgery, 42 (29%) received whole brain radiation, and 47 (33%) received stereotactic radiation. The median OS was 15 months, and a third of patients were alive at 2 years. The toxicity of ICIs was at 28% and 15% for Common Terminology Criteria for Adverse Events grade 1-2 and 3-4 events, respectively. Of the patients who are still alive, 76% of patients remained symptomatic neurologically at last follow-up. CONCLUSION: Most patients in this NZ real-world study were symptomatic and received anti-PD1 monotherapy. Approximately one-third of treated patients are alive at 2 years, but most patients remained symptomatic. This highlights the need for more effective treatment and prospective management of their neurologic rehabilitation needs.

The Effect of Process Parameters on the Microstructure, Stability, and Sensorial Properties of an Emulsion Cream Formulation.

A classical emulsion formulation based on petrolatum and mineral oil as the internal phase with emulsifier wax as a typical topical emulsion cream was investigated for the effect of process parameters on drug product quality and performance attributes. The Initial Design of Experiment (DoE) suggested that an oil phase above 15%, coupled with less than 10% emulsifying wax, resulted in less stable emulsions. Different processing parameters such as homogenization speed, duration, cooling rate, and final temperature showed minimal influence on properties and failed to improve stability. The final DoE suggested that the optimal emulsion stability was achieved by introducing a holding period midway through the cooling stage after solvent addition. Within the studied holding temperature range (25-35 °C), a higher holding temperature correlated with increased emulsion stability. However, the application of shear during the holding period, using a paddle mixer, adversely affected stability by disrupting the emulsion microstructure. IVRT studies revealed that the release of lidocaine was higher in the most stable emulsion produced at a holding temperature of 35 °C compared to the least stable emulsion produced at a holding temperature of 25 °C. This suggests that a holding temperature of 35 °C improves both the stability and active release performance. It appears that a slightly higher holding temperature, 35 °C, allows a more flexible and stable emulsifying agent film around the droplets facilitating stabilization of the emulsion. This study offers valuable insights into the relationship between process parameters at various stages of manufacture, microstructure, and various quality attributes of emulsion cream systems. The knowledge gained will facilitate improved design and optimization of robust manufacturing processes, ensuring the production of the formulations with the desired critical quality attributes.

Influence of Manufacturing Process on the Microstructure, Stability, and Sensorial Properties of a Topical Ointment Formulation.

The manufacturing process for ointments typically involves a series of heating, cooling, and mixing steps. Precise control of the level of mixing through homogenization and the cooling rate, as well as temperature at different stages, is important in delivering ointments with the desired quality attributes, stability, and performance. In this work, we investigated the influence of typical plant processing conditions on the microstructure, stability, and sensorial properties of a model ointment system through a Design of Experiments (DoE) approach. Homogenization speed at the cooling stage after the addition of the solvent (propylene glycol, PG) was found to be the critical processing parameter that affects stability and the rheological and sensorial properties of the ointment. A lower PG addition temperature was also found to be beneficial. The stabilization of the ointment at a lower PG addition temperature was hypothesized to be due to more effective encapsulation by crystallizing mono- and diglycerides at the lower temperature. The in vitro release profiles were found to be not influenced by the processing parameters, suggesting that for the ointment platform studied, processing affects the microstructure, but the effects do not translate into the release profile, a key performance indicator. Our systematic study represents a Quality-by-Design (QbD) approach to the design of a robust manufacturing process for delivering stable ointments with the desired performance attributes and properties.

5S clavam biosynthesis is controlled by an atypical two-component regulatory system in Streptomyces clavuligerus.

Streptomyces clavuligerus produces a collection of five clavam metabolites, including the clinically important ß-lactamase inhibitor clavulanic acid, as well as four structurally related metabolites called 5S clavams. The paralogue gene cluster of S. clavuligerus is one of three clusters of genes for the production of these clavam metabolites. A region downstream of the cluster was analyzed, and snk, res1, and res2, encoding elements of an atypical two-component regulatory system, were located. Mutation of any one of the three genes had no effect on clavulanic acid production, but snk and res2 mutants produced no 5S clavams, whereas res1 mutants overproduced 5S clavams. Reverse transcriptase PCR analyses showed that transcription of cvm7p (which encodes a transcriptional activator of 5S clavam biosynthesis) and 5S clavam biosynthetic genes was eliminated in snk and in res2 mutants but that snk and res2 transcription was unaffected in a cvm7p mutant. Both snk and res2 mutants could be complemented by introduction of cvm7p under the control of an independently regulated promoter. In vitro assays showed that Snk can autophosphorylate and transfer its phosphate group to both Res1 and Res2, and Snk-H365, Res1-D52, and Res2-D52 were identified as the phosphorylation sites for the system. Dephosphorylation assays indicated that Res1 stimulates dephosphorylation of Res2∼P. These results suggest a regulatory cascade in which Snk and Res2 form a two-component system controlling cvm7p transcription, with Res1 serving as a checkpoint to modulate phosphorylation levels. Cvm7P then activates transcription of 5S clavam biosynthetic genes.

Pembrolizumab-induced Myasthenia Gravis: Literature Review of Ocular Manifestations and a Refractory Case.

Immune checkpoint inhibition is a new and promising therapy approved for the treatment of various malignancies. Pembrolizumab is a potent tumor suppressor that acts by upregulating the immune system to recognize cancer cells which may result in disrupted self-tolerance. We describe a case and perform a literature review of myasthenia gravis with ocular manifestations after treatment with pembrolizumab. Our case had bilateral ptosis refractory to conventional treatment, and she remained functionally blind as a result. The literature review included 28 cases of immune-related myasthenia gravis, and a 30% mortality rate excluding deaths from primary cancer progression was shown. Under half had full symptom resolution (n=13, 46%), and there was no clear correlation between specific management strategies and prognosis. Patients with isolated ocular myasthenia gravis (n=9, 32%) were twice as likely to be symptom-free after treatment compared with generalized myasthenia gravis (75% vs. 39%). Respiratory involvement was associated with twice the mortality rate (60% vs. 33%) and triple the risk of noncomplete symptom resolution (20% vs. 61%). The majority of cases had their pembrolizumab discontinued (n=20, 71%), but 3 were successfully rechallenged by utilizing prophylactic low-dose steroids. Patients with immune-related myasthenia gravis experience increased mortality and morbidity but if steroid-responsive, may benefit from the reintroduction of anti-programmed cell death protein 1 therapy for end-stage malignancy with close monitoring. A high index of clinical suspicion for immune-related adverse effects are critical in an era of rising immunotherapy use.

Development and pilot-testing of a Decision Aid for use among Chinese women facing breast cancer surgery.

BACKGROUND: Women choosing breast cancer surgery encounter treatment decision-making (TDM) difficulties, which can cause psychological distress. Decision Aids (DAs) may facilitate TDM, but there are no DAs designed for Chinese populations. We developed a DA for Chinese women newly diagnosed with breast cancer, for use during the initial surgical consultation. AIMS: Conduct a pilot study to assess the DA acceptability and utility among Chinese women diagnosed with breast cancer. METHODS: Women preferred the DA in booklet format. A booklet was developed and revised and evaluated in two consecutive pilot studies (P1 and P2). On concluding their initial diagnostic consultation, 95 and 38 Chinese women newly diagnosed with breast cancer received the draft and revised draft DA booklet, respectively. Four-day post-consultation, women had questionnaires read out to them and to which they responded assessing attitudes towards the DA and their understanding of treatment options. RESULTS: The original DA was read/partially read by 66/22% (n = 84) of women, whilst the revised version was read/partially read by 74/16% (n = 35), including subliterate women (χ(2) = 0.76, P = 0.679). Knowledge scores varied with the extent the booklet was read (P1: F = 12.68, d.f. 2, P < 0.001; P2: F = 3.744, d.f. 2, P = 0.034). The revised, shorter version was graphically rich and resulted in improved perceived utility, [except for the 'treatment options' (χ(2) = 5.50, P = 0.019) and 'TDM guidance' (χ(2) = 8.19, P = 0.004) sections] without increasing anxiety (F = 0.689, P = 0.408; F = 3.45, P = 0.073). CONCLUSION: The DA was perceived as acceptable and useful for most women. The DA effectiveness is currently being evaluated using a randomized controlled trial.

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2- γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.See related Spotlight on p. 600.

Stabilization of ferulic acid in topical gel formulation via nanoencapsulation and pH optimization.

Ferulic acid is a potent anti-oxidant with scientifically proven skin care efficacies. However, instability of this active in the skin care products restricted its wide application in beauty and skin care industries. This study aimed to stabilize ferulic acid in topical hydrogel formulation via nanoencapsulation technique. Ferulic acid loaded nanocapsules were prepared via high pressure homogenization method and physicochemically characterized. Mean particle size of ferulic acid loaded nanocapsules was < 300 nm. TEM and SEM images exhibited spherical particles with smooth surface. DSC and XRD results indicated that ferulic acid was completely dissolved in the lipid matrix of the nanocapsules and remained in amorphous form. Two types of hydrogel formulations containing ferulic acid loaded nanocapsules were prepared: Gel A with pH higher and Gel B with pH lower than pKa of ferulic acid. Cross-polarized microscopic image of the gel formulations did not show presence of any un-encapsulated and un-dissolved crystal. Gel B showed slower and controlled release of ferulic acid than Gel A. Ferulic acid permeation through skin mimic from the gel formulation demonstrated controlled permeation. Color stability of the gel and chemical stability of ferulic acid were very good in Gel B, while poor in Gel A (although significantly better than the gel with un-encapsulated ferulic acid). The result clearly indicates that together with nanoencapsulation, low pH (less than pKa of ferulic acid) of the hydrogel was crucial for both product appearance and chemical stability of ferulic acid. In fact, it has been proved that skin care product with low pH is good for skin as it can maintain skin homeostasis and microbiome. Furthermore, the permeation result suggests that ferulic acid may penetrate into deep skin layers and at the same time avoid systemic circulation. Overall, this low pH hydrogel formulation containing nanoencapsulated ferulic acid demonstrates great promise for commercialization.

18F-FDG PET/CT based spleen to liver ratio associates with clinical outcome to ipilimumab in patients with metastatic melanoma.

BACKGROUND: Immune checkpoint blockade such as ipilimumab and anti-PD1 monoclonal antibodies have significantly improved survival in advanced melanoma. Biomarkers are urgently needed as a majority of patients do not respond, despite treatment-related toxicities. We analysed pre-treatment 18F-fluorodeoxyglucose positron emission tomography/computerised tomography (FDG PET/CT) parameters to assess its correlation with patient outcome. METHODS: This retrospective study evaluated pre-treatment FDG PET/CT scans in a discovery cohort of patients with advanced melanoma treated with ipilimumab or anti-PD1. Pre-treatment scans were assessed for maximum tumoral standardised uptake value (SUVmax), metabolic tumour volume (MTV) and spleen to liver ratio (SLR). Progression-free survival (PFS) and overall survival (OS) were characterised and modelled using univariable and multivariable analyses. Correlation of SLR and OS was validated in an independent cohort. Blood parameters and stored sera of patients from the discovery cohort was analysed to investigate biological correlates with SLR. RESULTS: Of the 90 evaluable patients in the discovery cohort: 50 received ipilimumab monotherapy, 20 received anti-PD1 monotherapy, and 20 patients received ipilimumab followed by anti-PD1 upon disease progression. High SLR > 1.1 was associated with poor PFS (median 1 vs 3 months; HR 3.14, p = 0.008) for patients treated with ipilimumab. High SLR was associated with poor OS after ipilimumab (median 1 vs 21 months; HR 5.83, p = 0.0001); as well as poor OS after first line immunotherapy of either ipilimumab or anti-PD1 (median 1 vs 14 months; HR 3.92, p = 0.003). The association of high SLR and poor OS after ipilimumab was validated in an independent cohort of 110 patients (median 2.3 months versus 11.9 months, HR 3.74). SLR was associated with poor OS in a multi-variable model independent of stage, LDH, absolute lymphocyte count and MTV. CONCLUSIONS: Pre-treatment Spleen to liver ratio (SLR) > 1.1 was associated with poor outcome after ipilimumab in advanced melanoma. This parameter warrants prospective evaluation.

Development of a scoring system from noncontrast computerized tomography measurements to improve the selection of upper ureteral stone for extracorporeal shock wave lithotripsy.

PURPOSE: We investigated the role of noncontrast computerized tomography in predicting the treatment outcome of shock wave lithotripsy on upper ureteral stones to formulate a clinical algorithm to facilitate clinical management. MATERIALS AND METHODS: Adult patients with upper ureteral stones confirmed by noncontrast computerized tomography and scheduled for primary in situ shock wave lithotripsy were prospectively recruited. Standardized treatment was performed on each patient. The primary end point was stone-free status at 3 months. Pretreatment noncontrast computerized tomography was assessed by a single radiologist blinded to the clinical parameters. Predictive values of computerized tomography measurements on the treatment outcome were then assessed. RESULTS: Between October 2004 and July 2007 a total of 94 patients (60 male and 34 female) were recruited for the study. Logistic regression showed that stone volume, mean stone density and skin-to-stone distance were potential predictors of successful treatment. From ROC curves the optimum cutoff for predicting treatment outcomes for stone volume, mean stone density and skin-to-stone distance was 0.2 cc, 593 HU and 9.2 cm, respectively. A simple scoring system was constructed based on the 3 factors of stone volume less than 0.2 cc, mean stone density less than 593 HU or skin-to-stone distance less than 9.2 cm. The stone-free rate for patients having 0, 1, 2 and 3 factors was 17.9%, 48.4%, 73.3% and 100%, respectively (linear-by-linear association test 22.83, p <0.001). CONCLUSIONS: Stone volume, mean stone density and skin-to-stone distance were potential predictors of the successful treatment of upper ureteral stones with shock wave lithotripsy. A scoring system based on these 3 factors helps separate patients into outcome groups and facilitates treatment planning.

Risk factors for and timing of death of extremely preterm infants.

This study examined risk factors for timing and cause of death for extremely preterm infants > or = 23 weeks and < 28 weeks. There were 479 liveborn infants and 98 deaths reviewed over a ten-year period. Thirty-two deaths (33%) occurred on the first day of life and 72 (75%) in the first month of life. Lower gestation and intrauterine growth restriction were significant risk factors for death. Most deaths occurred in the first month of life and at the lowest gestation in the first week.

Effect of vardenafil on blood pressure profile of patients with erectile dysfunction concomitantly treated with doxazosin gastrointestinal therapeutic system for benign prostatic hyperplasia.

PURPOSE: We investigated the effect of the combination of the doxazosin gastrointestinal therapeutic system and 10 mg vardenafil on the hemodynamic status of patients with benign prostatic hyperplasia and erectile dysfunction. MATERIALS AND METHODS: This was a double-blinded, randomized, placebo controlled crossover trial. Patients with benign prostatic hyperplasia and erectile dysfunction treated with the doxazosin gastrointestinal therapeutic system on a regular basis, with no other antihypertensive events, were recruited. Subjects took 10 mg vardenafil or placebo in a randomized crossover fashion with a washout period of at least 7 days between each treatment. The supine and standing blood pressure of the subjects was recorded from 1 hour before to 6 hours after the administration of vardenafil or placebo. The primary outcome of the study was the maximal change in standing systolic blood pressure of the subjects from 1 half hour before to 6 hours after the administration of drugs. RESULTS: A total of 37 patients, 25 (67.6%) and 12 (32.4%) on the doxazosin gastrointestinal therapeutic system at 4 mg and 8 mg, respectively, completed the trial. The combination drug therapy resulted in a maximal decrease in standing systolic blood pressure of 6.18 mm Hg (95% CI -12.02, -0.33; p = 0.039). Only 1 patient had an asymptomatic standing systolic blood pressure of less than 85 mm Hg. Otherwise no symptomatic hypotension or clinically significant adverse cardiovascular event was observed during the study. CONCLUSIONS: In patients on the doxazosin gastrointestinal therapeutic system for benign prostatic hyperplasia a single 10 mg dose of vardenafil had no symptomatic hemodynamic effects.

5S clavam biosynthetic genes are located in both the clavam and paralog gene clusters in Streptomyces clavuligerus.

The Streptomyces clavuligerus clavam gene cluster was examined to identify genes specifically involved in 5S clavam biosynthesis. A reduction/loss of 5S clavam production was seen in cvm2 and cvm5 gene mutants, and a clavam metabolite not previously observed, 2-carboxymethylideneclavam, accumulated in the cvm5 mutant. Disruption of additional genes from the region of the clavam cluster did not have any effect on 5S clavam production. Examination of the paralog gene cluster region for 5S clavam biosynthetic genes led to the identification of cvm6P and cvm7P, which encode a putative aminotransferase and a transcriptional regulator, respectively. Mutants defective in cvm6P and cvm7P were completely blocked in 5S clavam but not clavulanic acid production. The loss of 5S clavam production in cvm7P mutants suggests that this gene encodes a transcriptional regulator specific for 5S clavam metabolite biosynthesis.