Rational Design and Multicomponent Synthesis of Lipid-Peptoid Nanocomposites towards a Customized Drug Delivery System Assembly.

Nanotechnology has assumed a significant role over the last decade in the development of various technologies applied to health sciences. This becomes even more evident with its application in controlled drug delivery systems. In this context, peptoids are a promising class of compounds for application as nanocarriers in drug delivery systems. These compounds can be obtained efficiently and with highly functionalized structural diversity via the Ugi 4-component reaction (U-4CR). Herein, we report the design of the process control strategy for the future development of lipid-peptoid-based customized drug delivery system assemblies. Over 20 lipid-peptoid nanocomposites were synthesized via the U-4CR in good to excellent yields. These products were successfully submitted to the nanoparticle formation by the emulsification-evaporation process from lipophilic solution and analyzed via Dynamic Light Scattering (DLS). Several molecules generated nanoparticles with a size ≤200 nm, making them good candidates for drug delivery systems, such as in cancer treatment.

Identification of somatic gene mutations in penile squamous cell carcinoma.

There is a lack of studies on somatic gene mutations and cell signaling driving penile carcinogenesis. Our objective was to analyze somatic mutations in genes downstream of EGFR in penile squamous cell carcinomas, especially the mTOR and RAS/MAPK pathways. We retrospectively analyzed somatic mutations in 10 in situ and 65 invasive penile squamous cell carcinomas by using Sequenom's Mass Spectrometry iPlex Technology and Oncocarta v1.0 Panel. The DNA was extracted from FFPE blocks and we identified somatic missense mutations in three in situ tumors and in 19 invasive tumors, mostly in PIK3CA, KRAS, HRAS, NRAS, and PDGFA genes. Somatic mutations in the PIK3CA gene or RAS family genes were neither associated with tumor grade, stage or outcome, and were equally often identified in hrHPV positive and in hrHPV negative tumors that showed no p53 expression. Mutations in PIK3CA, KRAS, and HRAS are frequent in penile squamous cell carcinoma and likely play a role in the development of p53-negative tumors. Although the presence of these mutations does not seem to correlate with tumoral behavior or outcome, they could be biomarkers of treatment failure with anti-EGFR mAb in patients with penile squamous cell carcinoma.

A sequential Ugi multicomponent/Cu-catalyzed azide-alkyne cycloaddition approach for the continuous flow generation of cyclic peptoids.

The development of a continuous flow multistep strategy for the synthesis of linear peptoids and their subsequent macrocyclization via Click chemistry is described. The central transformation of this process is an Ugi four-component reaction generating the peptidomimetic core structure. In order to avoid exposure to the often toxic and malodorous isocyanide building blocks, the continuous approach was telescoped by the dehydration of the corresponding formamide. In a concurrent operation, the highly energetic azide moiety required for the subsequent intramolecular copper-catalyzed azide-alkyne cycloaddition (Click reaction) was installed by nucleophilic substitution from a bromide precursor. All steps yielding to the linear core structures can be conveniently coupled without the need for purification steps resulting in a single process generating the desired peptidomimetics in good to excellent yields within a 25 min reaction time. The following macrocyclization was realized in a coil reactor made of copper without any additional additive. A careful process intensification study demonstrated that this transformation occurs quantitatively within 25 min at 140 °C. Depending on the resulting ring strain, either a dimeric or a monomeric form of the cyclic product was obtained.

Identification and genotyping of human papillomavirus in a Spanish cohort of penile squamous cell carcinomas: correlation with pathologic subtypes, p16(INK4a) expression, and prognosis.

BACKGROUND: Penile squamous cell carcinoma (PSCC) is a tumor with a high metastatic potential. In PSCC the attributable fraction to human papillomavirus (HPV) is not well established. OBJECTIVE: We sought to provide novel data about the prevalence of HPV in a large series of penile intraepithelial neoplasia (PeIN) and invasive PSCC, correlating the results with the histologic subtype, p16(INK4a) immunostaining, and prognosis. METHODS: A total of 82 PSCC were included in the study, 69 invasive and 13 PeIN. HPV detection was performed by polymerase chain reaction with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. P16(INK4a) immunohistochemical expression on tissue microarrays was also analyzed. RESULTS: HPV DNA was identified in 31 of 77 (40.2%) PSCC (22 of 67 invasive and 9 of 10 PeIN). In 25 of 31 (80.6%) cases HPV-16 was identified. HPV detection was significantly associated with some histologic subtypes: most basaloid and warty tumors were high-risk HPV (hrHPV) positive, whereas only 15% of usual PSCC were hr-HPV positive. All hrHPV-positive PSCC had an adjacent undifferentiated PeIN. Strong p16(INK4a) immunostaining correlated with hrHPV infection. Most undifferentiated PeIN showed p16(INK4a) immunohistochemical overexpression. Both hrHPV-positive and p16(INK4a)-positive tumors showed a better overall survival without reaching statistical significance. LIMITATIONS: This was a retrospective study. CONCLUSIONS: Our results suggest that most hrHPV-positive PSCC develop from undifferentiated hrHPV-positive PeIN. P16(INK4a) immunostaining may be useful in identifying both etiologically related hrHPV-positive tumors and those with better outcome. The routine use of p16(INK4a) staining should be incorporated in histologic evaluation of PSCC.

Heart failure in primary care: co-morbidity and utilization of health care resources.

BACKGROUND: In order to ensure proper management of primary care (PC) services, the efficiency of the health professionals tasked with such services must be known. Patients with heart failure (HF) are characterized by advanced age, high co-morbidity and high resource utilization. OBJECTIVE: To ascertain PC resource utilization by HF patients and variability in the management of such patients by GPs. METHODS: Descriptive, cross-sectional study targeting a population attended by 129 GPs over the course of 1 year. All patients with diagnosis of HF in their clinical histories were included, classified using the Adjusted Clinical Group system and then grouped into six resource utilization bands (RUBs). Resource utilization and Efficiency Index were both calculated. RESULTS: One hundred per cent of patients with HF were ranked in RUBs 3, 4 and 5. The highest GP visit rate was 20 and the lowest in excess of 10 visits per year. Prescription drug costs for these patients ranged from €885 to €1422 per patient per year. Health professional efficiency varied notably, even after adjustment for co-morbidity (Efficiency Index Variation Ratio of 28.27 for visits and 404.29 for prescription drug cost). CONCLUSIONS: Patients with HF register a high utilization of resources, and there is great variability in the management of such patients by health professionals, which cannot be accounted for by the degree of case complexity.

Comorbidity in patients with chronic obstructive pulmonary disease in family practice: a cross sectional study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is frequent and often coexists with other diseases. The aim of this study was to quantify the prevalence of COPD and related chronic comorbidity among patients aged over 40 years visiting family practices in an area of Madrid. METHODS: An observational, descriptive, cross-sectional study was conducted in a health area of the Madrid Autonomous Region (Comunidad Autónoma de Madrid). The practice population totalled 198,670 persons attended by 129 Family Physicians (FPs), and the study population was made up of persons over the age of 40 years drawn from this practice population. Patients were deemed to have COPD if this diagnosis appeared on their clinical histories. Prevalence of COPD; prevalence of a further 25 chronic diseases in patients with COPD; and standardised prevalence ratios, were calculated. RESULTS: Prevalence of COPD in family medicine was 3.2% (95% CI 3.0-3.3) overall, 5.3% among men and 1.4% among women; 90% of patients presented with comorbidity, with a mean of 4 ± 2.04 chronic diseases per patient, with the most prevalent related diseases being arterial hypertension (52%), disorders of lipid metabolism (34%), obesity (25%), diabetes (20%) and arrhythmia (15%). After controlling for age and sex, the observed prevalence of the following ten chronic diseases was higher than expected: heart failure; chronic liver disease; asthma; generalised artherosclerosis; osteoporosis; ischaemic heart disease; thyroid disease; anxiety/depression; arrhythmia; and obesity. CONCLUSIONS: Patients with COPD, who are frequent in family practice, have a complex profile and pose a clinical and organisational challenge to FPs.

Dissolved oxygen regulation by logarithmic/antilogarithmic control to improve a wastewater treatment process.

This paper presents the automation of a real activated sludge wastewater treatment plant, which is located at San Antonio Ajijic in Jalisco, Mexico. The main objective is to create an on-line automatic supervision system, and to regulate the dissolved oxygen concentration in order to improve the performances of the process treating municipal wastewater. An approximate mathematical model is determined in order to evaluate via simulations different control strategies: proportional integral (PI), fuzzy PI and PI Logarithm/Antilogarithm (PI L/A). The controlled variable is dissolved oxygen and the control input is the injected oxygen. Based on this evaluation, the PI L/A controller is selected to be implemented in the real process. After that, the implementation, testing and fully operation of the plant automation are described. With this system, the considered wastewater treatment plant save energy and improves the effluent quality; also, the process monitoring is done online and it is easily operated by the plant users.

Atherosclerotic-Derived Endothelial Cell Response Conducted by Titanium Oxide Nanotubes.

Atherosclerosis lesions are described as the formation of an occlusive wall-vessel plaque that can exacerbate infarctions, strokes, and even death. Furthermore, atherosclerosis damages the endothelium integrity, avoiding proper regeneration after stent implantation. Therefore, we investigate the beneficial effects of TiO2 nanotubes (NTs) in promoting the initial response of detrimental human atherosclerotic-derived endothelial cells (AThEC). We synthesized and characterized NTs on Ti6Al4V by anodization. We isolated AThEC and tested the adhesion long-lasting proliferation activity, and the modulation of focal adhesions conducted on the materials. Moreover, ultrastructural cell-surface contact at the nanoscale and membrane roughness were evaluated to explain the results. Our findings depicted improved filopodia and focal adhesions stimulated by the NTs. Similarly, the NTs harbored long-lasting proliferative metabolism after 5 days, explained by overcoming cell-contact interactions at the nanoscale. Furthermore, the senescent activity detected in the AThEC could be mitigated by the modified membrane roughness and cellular stretch orchestrated by the NTs. Importantly, the NTs stimulate the initial endothelial anchorage and metabolic recovery required to regenerate the endothelial monolayer. Despite the dysfunctional status of the AThEC, our study brings new evidence for the potential application of nano-configured biomaterials for innovation in stent technologies.

Observation versus antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia.

The effectiveness of antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia (ET) is not proven. In this study, the incidence rates of arterial and venous thrombosis were retrospectively analyzed in 300 low-risk patients with ET treated with antiplatelet drugs as monotherapy (n = 198) or followed with careful observation (n = 102). Follow-up was 802 and 848 person-years for antiplatelet therapy and observation, respectively. Rates of thrombotic events were 21.2 and 17.7 per 1000 person-years for antiplatelet therapy and observation, respectively (P = .6). JAK2 V617F-positive patients not receiving antiplatelet medication showed an increased risk of venous thrombosis (incidence rate ratio [IRR]: 4.0; 95% CI: 1.2-12.9; P = .02). Patients with cardiovascular risk factors had increased rates of arterial thrombosis while on observation (IRR: 2.5; 95% CI: 1.02-6.1; P = .047). An increased risk of major bleeding was observed in patients with platelet count greater than 1000 x 10(9)/L under antiplatelet therapy (IRR: 5.4; 95% CI: 1.7-17.2; P = .004). In conclusion, antiplatelet therapy reduces the incidence of venous thrombosis in patients with JAK2-positive ET and the rate of arterial thrombosis in patients with associated cardiovascular risk factors. In the remaining low-risk patients, this therapy is not effective as primary prophylaxis of thrombosis, and observation may be an adequate option.

Combining chemometric and phytochemical tools to isolate and characterize activity of Vismia gracilis compounds against Aedes aegypti.

Vismia gracilis extracts were tested against Aedes aegypti to assess mortality and behavioural effects. The leaf hexanic extract (L-Hex) presented increased larvicidal activity with exposure period: LC50 46.48 µg/mL (24 h) and LC50 20.57 µg/mL (48 h). Eight compounds were annotated/isolated from the L-Hex active extract, 4 benzophenones and 4 anthraquinones. Considering chemometric findings, the benzophenone moiety, tested as the commercial benzophenone, promoted larval mortality (LC50 16.35 µg/mL). Both the L-Hex extract and benzophenone induced intestinal damage in larvae. Benzophenone also promoted toxicity and behavioural effects in female adults. These findings highlighted the potential use of this class of compounds for developing vector-control products.

Lenalidomide-dexamethasone versus observation in high-risk smoldering myeloma after 12 years of median follow-up time: A randomized, open-label study.

BACKGROUND: Smoldering multiple myeloma (SMM) is a heterogeneous disease in terms of progression to myeloma (MM), but its standard of care continues to be observation. METHODS: The QuiRedex phase 3 trial initiated in 2007 included 119 high-risk patients with SMM randomized to treatment or observation. Treatment consisted of nine 4-week induction cycles (lenalidomide [Rd], 25 mg on days 1-21 plus dexamethasone, 20 mg on days 1-4 and 12-15), followed by maintenance (R, 10 mg on days 1-21) for up to 2 years. The primary end-point was time to progression (TTP) to myeloma based on per protocol population. Secondary end-points were overall survival (OS), response rate, and safety. An update of the trial after a long-term follow-up is presented here. This trial was registered with ClinicalTrials.gov (NCT00480363). FINDINGS: After a median follow-up time of 12.5 years (range: 10.4-13.6), the median TTP to MM was 2.1 years in the observation arm and 9.5 years in the Rd arm (HR: 0.28, 95% CI: 0.18-0.44, p < 0.0001). The median OS was 8.5 years in the abstention arm and not reached in the Rd group (HR: 0.57, 95% CI: 0.34-0.95, p = 0.032). Patients who progressed received optimized treatments according to the standards of care, and the OS from progression was comparable in both arms (p = 0.96). INTERPRETATION: This analysis confirms that early treatment with Rd for high-risk SMM translates into a sustained benefit in both TTP and OS. FUNDING: Pethema (Spanish Program for the Treatment of Hematologic Diseases), Spain.

Heart failure in the family practice: a study of the prevalence and co-morbidity.

BACKGROUND: Heart failure (HF) is a health problem that particularly affects the elderly population. Its onset is associated with other chronic diseases, a circumstance that makes it a challenge for health care services. The aim of this study is to quantify the prevalence of HF in family medicine offices and describe the chronic co-morbidity associated with it. METHODS: A cross-sectional, observational descriptive study set in a health area of the Community of Madrid, Spain. The study was carried out in a population of 198,670 individuals over 14 years of age, attended to by 129 specialists in family medicine. The patient was considered to have HF when this diagnosis (ICPC code K77) appeared in his or her electronic medical record. The prevalence of HF was quantified and its association with another 25 chronic diseases was analysed. RESULTS: The prevalence of HF was 6.9‰, 7.9‰ among women and 5.9‰ among men. Patients with HF had a high rate of chronic co-morbidity, with an average of 5.2 + 2.1 chronic diseases. Only 3% of the patients present with isolated HF and >60% have four or more additional chronic problems. Hypertension, cardiac arrhythmias, hyperlipidaemia, obesity and diabetes mellitus are the chronic diseases most frequently detected in HF patients. CONCLUSION: Patients with HF frequently visit the offices of family physicians, presenting with a high rate of cardiac and non-cardiac co-morbidity that proves to be a challenge on the clinical level and in terms of the organization of health care services.

[Holmium laser enucleation of the prostatic adenoma]. / Enucleación del adenoma prostático mediante el láser de Holmio (HOLEP).

Holmium laser enucleation of the prostatic adenoma (HoLEP) represents an innovative surgical option for the treatment of bladder outlet obstruction caused by benign prostatic hypertrophy. The results of numerous randomized prospective studies and clinical case series have confirmed that HoLEP is a procedure that attains immediate bladder outlet obstruction release, that improvement of symptomatic and uroflowmetry parameters is maintained in the midterm and, it is associated with less morbidity than conventional surgery. On the other hand, the shortage of urologists with experience in this procedure, and its technical difficulty have limited its spread in our environment. In this article we describe in detail the technique we use in our center for the performance of HoLEP, emphasizing the modifications we have introduced with time to make the operation easier and to avoid complications.

Structure-activity relationship of diameter controlled Ag@Cu nanoparticles in broad-spectrum antibacterial mechanism.

Current outbreaks associated with drug-resistant clinical strains are demanding for the development of broad-spectrum antibacterial agents. The bactericidal materials should be eco-friendly, economical and effective to suppress bacterial growth. Thus, in this work, diameter controlled spherical Cucore-Agshell nanoparticles (Ag@CuNPs) with diameter ranging from 70 to 100 nm by one-step co-reduction approach were designed and synthesized. The Ag@CuNPs were homogenous, stable, and positively charged. The 70 nm Ag@CuNPs showed a consistent and regular Ag shielding. We observed the 100 nm Ag@CuNPs achieved symmetrical doped Ag clusters on the Cu core surface. We used Gram-positive and Gram-negative models strains to test the wide-spectrum antibacterial activity. The Ag@CuNPs showed detrimental microbial viability in a dose-dependent manner; however, 70 nm Ag@CuNPs were superior to those of 100 nm Ag@CuNPs. Initially, Ag@CuNPs attached and translocated the membrane surface resulting in bacterial eradication. Our analyses exhibited that antibacterial mechanism was not governed by the bacterial genre, nonetheless, by cell type, morphology, growing ability and the NPs uptake capability. The Ag@CuNPs were highly tolerated by human fibroblasts, mainly by the use of starch as glucosidic capper and stabilizer, suggesting optimal biocompatibility and activity. The Ag@CuNPs open up a novel platform to study the potential action of bimetallic nanoparticles and their molecular role for biomedical, clinical, hospital and industrial-chemical applications.

Promotion of Surgical Masks Antimicrobial Activity by Disinfection and Impregnation with Disinfectant Silver Nanoparticles.

BACKGROUND: The COVID-19 pandemic is requesting highly effective protective personnel equipment, mainly for healthcare professionals. However, the current demand has exceeded the supply chain and, consequently, shortage of essential medical materials, such as surgical masks. Due to these alarming limitations, it is crucial to develop effective means of disinfection, reusing, and thereby applying antimicrobial shielding protection to the clinical supplies. PURPOSE: Therefore, in this work, we developed a novel, economical, and straightforward approach to promote antimicrobial activity to surgical masks by impregnating silver nanoparticles (AgNPs). METHODS: Our strategy consisted of fabricating a new alcohol disinfectant formulation combining special surfactants and AgNPs, which is demonstrated to be extensively effective against a broad number of microbial surrogates of SARS-CoV-2. RESULTS: The present nano-formula reported a superior microbial reduction of 99.999% against a wide number of microorganisms. Furthermore, the enveloped H5N1 virus was wholly inactivated after 15 min of disinfection. Far more attractive, the current method for reusing surgical masks did not show outcomes of detrimental amendments, suggesting that the protocol does not alter the filtration effectiveness. CONCLUSION: The nano-disinfectant provides a valuable strategy for effective decontamination, reuse, and even antimicrobial promotion to surgical masks for frontline clinical personnel.

Azadirachta indica Leaf Extract as Green Corrosion Inhibitor for Reinforced Concrete Structures: Corrosion Effectiveness against Commercial Corrosion Inhibitors and Concrete Integrity.

The construction industry has extensively demanded novel green inhibition strategies for the conservation and protection of carbon steel-reinforced concrete structures. For the first time, the effect of Azadirachta indica leaf extract (Neem) as a potential corrosion inhibitor of carbon steel in reinforced concrete under corrosion in saline simulated media was evaluated. To assess the corrosion inhibition behavior of the Neem natural organic extract, three inorganic commercial inhibitors were tested to compare following the criteria established by Stratful for half-cell potential under a simulated chloride environment. Moreover, the effect of concrete integrity by the Neem treatment was recorded after different temperature conditions, slump, weight alteration, air content, compressive strength, and chloride-ions penetration. The results suggested that the Neem treatments did not alter the concrete integrity and the physicochemical parameters. We reached a promoted long-term corrosion protection of 95% after 182 days of evaluation. Thus far, our current results open up a new promising "green" road to the conservation of carbon steel in reinforced concrete for the construction industry.

Analysis of the Micro-RNA-133 and PITX3 genes in Parkinson's disease.

MicroRNAs are small RNA sequences that negatively regulate gene expression by binding to the 3' untranslated regions of mRNAs. MiR-133b has been implicated in Parkinson's disease (PD) by a mechanism that involves the regulation of the transcription factor PITX3. The variation in these genes could contribute to the risk of developing PD. We searched for DNA variants in miR-133 and PITX3 genes in PD patients and healthy controls from Spain. We found common DNA variants in the three miR-133 genes. Genotyping of a first set of patients (n = 777) and controls (n = 650) showed a higher frequency of homozygous for a miR-133b variant (-90 del A) in PD-patients (6/575; 1%) than in healthy controls (0/650) (P = 0.03). However, this association was not confirmed in a second set of patients (1/250; 0.4%) and controls (2/210; 1%). No common PITX3 variants were associated with PD, although a rare missense change (G32S) was found in only one patient and none of the controls. In conclusion, we report the variation in genes of a pathway that has been involved in dopaminergic neuron differentiation and survival. Our work suggests that miR-133 and PITX3 gene variants did not contribute to the risk for PD.

Lrrk2 R1441G-related Parkinson's disease: evidence of a common founding event in the seventh century in Northern Spain.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene together represent the most common genetic determinant of Parkinson's disease (PD) identified to date. The vast majority of patients with LRRK2-related PD reported in the literature carry one of three pathogenic substitutions: G2019S, R1441C, or R1441G. While G2019S and R1441C are geographically widespread, R1441G is most prevalent in the Basque Country and is rare outside of Northern Spain. We sought to better understand the processes that have shaped the current distribution of R1441G. We performed a haplotype analysis of 29 unrelated PD patients heterozygous for R1441G and 85 wild-type controls using 20 markers that spanned 15.1 Mb across the LRRK2 region. Nine of the patients were of Basque origin and 20 were non-Basques. We inferred haplotypes using a Bayesian approach and utilized a maximum-likelihood method to estimate the age of the most recent common ancestor. Significant but incomplete allele sharing was observed over a distance of 6.0 Mb and a single, rare ten-marker haplotype 5.8 Mb in length was seen in all mutation carriers. We estimate that the most recent common ancestor lived 1,350 (95% CI, 1,020-1,740) years ago in approximately the seventh century. We hypothesize that R1441G originated in the Basque population and that dispersion of the mutation then occurred through short-range gene flow that was largely limited to nearby regions in Spain.

Mutational screening of the mortalin gene (HSPA9) in Parkinson's disease.

Mortalin is a mitochondrial chaperone of the heat shock protein 70 family. Mortalin plays a central role in mitochondrial biogenesis through its capacity to direct the import of nuclear-encoded proteins into the mitochondria. As mitochondrial dysfunction has been involved in Parkinson's disease (PD), changes in mortalin function and expression could manifest as a higher risk of developing PD. In agreement with this, mortalin expression was decreased in the mitochondrial fraction of neurons from the substantia nigra of PD patients. We hypothesised that DNA variants in the mortalin gene (HSPA9) could contribute to the risk of developing PD. We analysed the 17 HSPA9 coding exons in 330 PD patients and 250 controls. In addition to several polymorphisms, found in patients and controls, three variants were found in 3 patients but none of the controls: two missense (R126 > W and P509 > S) and a 17 bp insertion in intron 8 (predicted to affect RNA splicing). Our study suggests that putative mutations in the mortalin, although rare, could contribute to the risk of developing PD.

Individual variations of serum testosterone in patients with prostate cancer receiving androgen deprivation therapy.

OBJECTIVE: To analyse individual variations in serum testosterone level, the cumulative rate of 'breakthrough' increases over castrate levels, and to evaluate whether the increases can be predicted. PATIENTS AND METHODS: Serum testosterone levels were determined every 6 months over 3 years in 73 consecutive patients with prostate cancer who were medically castrated, prospectively enrolled in a single tertiary academic centre. Patients recruited for this study were being treated with a 3-monthly depot of luteinizing hormone-releasing hormone agonist over 6-48 months. Serum testosterone was measured using a chemiluminescent assay with a lower sensitivity level of 15 ng/dL and interassay coefficient of variation of 25% at low testosterone concentrations. RESULTS: Individual variations could not be explained by the interassay variation coefficient in 26% of the patients. The rate of breakthrough increases >50 ng/dL increased from 12.3% at the first determination to 24.7% at the third, then remaining stable. The rate of breakthrough increases of 20-50 ng/dL increased from 27.4% at the first determination to 31.5% at the second, and then remained stable. A first determination of <20 ng/dL provided an 11.4% probability for future increases of >50 ng/dL, with a 5.7% probability if two consecutive determinations were <20 ng/dL and a null probability when three consecutive determinations were <20 ng/dL. CONCLUSIONS: Individual variations in serum testosterone level cannot be explained by the coefficient of variation of the assay in a quarter of patients who are medically castrated. Breakthrough increases over castrate levels increase over time and those of >50 ng/dL can be predicted from the previous levels.