Small changes in thermal conditions hinder marathon running performance in the tropics.

We examined marathon performance of the same group of runners in relation to small changes in dry bulb temperature (Tdb) and wet bulb temperature (Twb) across 3 consecutive y, and investigated whether performance was poorer during an evening marathon compared with morning marathons. Marathon results were obtained from the 2017, 2018, and 2019 Standard Chartered Singapore Marathons. Tdb, Twb, Td, relative humidity, and absolute humidity were gathered for each marathon. K-means clustering and linear regressions were performed on 610 runners who participated in all three marathons. Analysis of the 610 runners' marathon performance was contrasted with Tdb and Twb. Linear regressions were also performed on 190 runners filtered by percentile, yielding similar results. For clusters with similar Tdb from all runners K-means clustering, an increase in mean Twb by 1.5°C coincided with an increase in finishing time by 559 s (9.3 min) (p < 0.033). Twb hinders marathon performance more than Tdb, with each percentage rise in Tdb and Twb resulting in an increase in net time by 7.6% and 39.1%, respectively (p < 0.025). Male and female runners' response to Tdb and Twb changes were similar (overlap in 95% confidence intervals for the respective regression coefficients). In conclusion, small variations in environmental parameters affected marathon performance, with Twb impairing marathon performance more than Tdb. Marathon performance was likely better in the morning than evening, possibly due to time of day differences, along with unfavorable Tdb that superseded training effects and the effects of lower Twb.

Whole-genome and transcriptome analysis enhances precision cancer treatment options.

BACKGROUND: Recent advances are enabling delivery of precision genomic medicine to cancer clinics. While the majority of approaches profile panels of selected genes or hotspot regions, comprehensive data provided by whole-genome and transcriptome sequencing and analysis (WGTA) present an opportunity to align a much larger proportion of patients to therapies. PATIENTS AND METHODS: Samples from 570 patients with advanced or metastatic cancer of diverse types enrolled in the Personalized OncoGenomics (POG) program underwent WGTA. DNA-based data, including mutations, copy number and mutation signatures, were combined with RNA-based data, including gene expression and fusions, to generate comprehensive WGTA profiles. A multidisciplinary molecular tumour board used WGTA profiles to identify and prioritize clinically actionable alterations and inform therapy. Patient responses to WGTA-informed therapies were collected. RESULTS: Clinically actionable targets were identified for 83% of patients, of which 37% of patients received WGTA-informed treatments. RNA expression data were particularly informative, contributing to 67% of WGTA-informed treatments; 25% of treatments were informed by RNA expression alone. Of a total 248 WGTA-informed treatments, 46% resulted in clinical benefit. RNA expression data were comparable to DNA-based mutation and copy number data in aligning to clinically beneficial treatments. Genome signatures also guided therapeutics including platinum, poly-ADP ribose polymerase inhibitors and immunotherapies. Patients accessed WGTA-informed treatments through clinical trials (19%), off-label use (35%) and as standard therapies (46%) including those which would not otherwise have been the next choice of therapy, demonstrating the utility of genomic information to direct use of chemotherapies as well as targeted therapies. CONCLUSIONS: Integrating RNA expression and genome data illuminated treatment options that resulted in 46% of treated patients experiencing positive clinical benefit, supporting the use of comprehensive WGTA profiling in clinical cancer care.

Visual serial dependence in an audiovisual stimulus.

Serial dependence is a phenomenon that biases the perception of features or objects systematically toward sensory input from the recent past (Fischer & Whitney, 2014). There is an active debate whether this effect is rooted directly in perception or reflects biases in decision making. We investigated serial dependence across three experiments by manipulating the decision made on each trial. A multimodal audiovisual stimulus comprising a Gabor and a vowel sound was presented repeatedly. On each trial, participants reported either the Gabor orientation or the vowel sound. Participants either ignored one modality (Experiment 1) or attended to both modalities (Experiments 2 and 3). In Experiments 2 and 3, the response task was randomized to prevent anticipating which modality to respond to until the response phase. In Experiment 3, no-response trials were additionally interleaved. Results across the three experiments demonstrated serial dependence only when participants reported the visual modality. Serial dependence was also present in visual reports when participants completed auditory reports or made no reports on previous trials. The previous stimulus alone was enough to elicit an effect. Serial dependence is unlikely to be an effect of the previous decision on the stimulus, but rather an effect of perceiving the previous stimulus.

Directional biases for blink adaptation in voluntary and reflexive eye blinks.

The oculomotor system is subject to noise, and adaptive processes compensate for consistent errors in gaze targeting. Recent evidence suggests that positional errors induced by eye blinks are also corrected by an adaptive process: When a fixation target is displaced during repeated blinks, subsequent blinks are accompanied by an automatic compensating eye movement anticipating the updated target location after the blink. Here, we further tested the extent of this "blink adaptation." Participants were tasked to look at a white target dot on a black screen and encouraged to blink voluntarily, or air puffs were used to elicit reflexive blinks. In separate runs, the target was displaced by 0.7° in either of the four cardinal directions during blinks. Participants adapted to positional changes during blinks, i.e., the postblink gaze position was biased in the direction of the dot displacement. Adaptation occurred for both voluntary and reflexive blinks. However, adaptation was unequal across different adaptation directions: Horizontally, temporal displacements experienced larger adaptation than nasal displacements; vertically, downward displacements led to larger adaptation than upward displacements. Results paralleled anisotropies commonly found for saccade amplitudes, and thus it is likely that gaze corrections across eye blinks share general constraints of the oculomotor system with saccades.

Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in ß-adrenoceptor signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation-contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38-/- mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38-/- myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of ß-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38-/- myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38-/- mice. Whole hearts isolated from CD38-/- mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of ß-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic ß-Adrenoceptor Signaling in the Heart.

Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of ß-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of ß-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute ß-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in ß-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of ß-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of ß-adrenoceptors to hypertrophy and associated arrhythmias.

Trends in Patterns of Posterior Uveitis and Panuveitis in a Tertiary Institution in Singapore.

PURPOSE: The study aims to analyze the trends of posterior uveitis and panuveitis patients seen by a tertiary eye center in Singapore between 2004 and 2012. METHODS: We conducted a retrospective analysis of 363 consecutive new cases of posterior uveitis and panuveitis. The cases were segregated into idiopathic, infectious, or noninfectious. RESULTS: We found statistically significant differences between etiologies and ethnicity (p = 0.014). We noticed a statistically significant downward trend (Spearman's rho (ρ) = -0.812, p = 0.008) for dengue uveitis, and an upward trend for the idiopathic category (Spearman's rho (ρ) = 0.753, p = 0.019). CONCLUSIONS: We observed differences between etiologies and ethnicity, pointing toward potential susceptibility variations. There was an upward trend of idiopathic causes, possibly due to better control of systemic and infectious etiologies. The dengue uveitis incidence correlates well with our national statistics. The downward trend of dengue uveitis could be due to the introduction of Singapore's dengue surveillance in 2005, emphasizing the importance of controlling the disease.

Characteristics of Cytomegalovirus Uveitis in Immunocompetent Patients.

PURPOSE: To present the clinical characteristics of patients with anterior uveitis who had evidence of cytomegalovirus (CMV) infection on polymerase chain reaction PCR-based assays for viral DNA in aqueous samples. METHODS: This was a retrospective observational case series of 16 patients with CMV infection on qualitative polymerase chain reaction PCR-based assays for viral DNA in aqueous samples. Case records of 16 patients were reviewed and relevant clinical information was collected using a standardized data sheet. RESULTS: There were 10 male and 6 female patients, with 16 eyes included. The median age at the first attack was 52 years (range 27-77 years). Thirteen patients (81.3%) presented with an initial BCVA of 20/40 or better. Eleven eyes (68.8%) had anterior chamber inflammation of 1+ cells or less. Eight eyes (50.0%) had concomitant sectoral iris atrophy, while 2 eyes were noted to have heterochromic irides. Eleven patients (68.8%) presented with an elevated intraocular pressure. Seven patients (43.8%) had clinical features that led to a presumptive diagnosis of Posner-Schlossman syndrome, while 3 patients (18.8%) were initially diagnosed with Fuchs heterochromic iridocyclitis. Six patients were initially treated for uveitic glaucoma or anterior uveitis of unknown cause. CONCLUSIONS: There is a spectrum of clinical manifestations of CMV anterior uveitis. A high index of suspicion of a possible viral etiology, especially CMV, and subsequent accurate identification of the virus involved are fundamental to the overall therapeutic approach.

Inhibition of angiotensin II-induced cardiac hypertrophy and associated ventricular arrhythmias by a p21 activated kinase 1 bioactive peptide.

Cardiac hypertrophy increases the risk of morbidity and mortality of cardiovascular disease and thus inhibiting such hypertrophy is beneficial. In the present study, we explored the effect of a bioactive peptide (PAP) on angiotensin II (Ang II)-induced hypertrophy and associated ventricular arrhythmias in in vitro and in vivo models. PAP enhances p21 activated kinase 1 (Pak1) activity by increasing the level of phosphorylated Pak1 in cultured neonatal rat ventricular myocytes (NRVMs). Such PAP-induced Pak1 activation is associated with a significant reduction of Ang II-induced hypertrophy in NRVMs and C57BL/6 mice, in vitro and in vivo, respectively. Furthermore, PAP antagonizes ventricular arrhythmias associated with Ang II-induced hypertrophy in mice. Its antiarrhythmic effect is likely to be involved in multiple mechanisms to affect both substrate and trigger of ventricular arrhythmogenesis. Thus our results suggest that Pak1 activation achieved by specific bioactive peptide represents a potential novel therapeutic strategy for cardiac hypertrophy and associated ventricular arrhythmias.

Trends in patterns of intermediate uveitis in a tertiary institution in Singapore.

PURPOSE: The study aims to describe the characteristics and etiologic causes of intermediate uveitis (IU) patients seen by a tertiary eye center in Singapore over 8 years. METHODS: This was a retrospective analysis of the clinical records of consecutive new cases of IU that presented to the uveitis subspecialty clinic from 2004-2011 at Tan Tock Seng Hospital. Data collected included demographics, clinical and laboratory findings. Diagnoses were based on standardized clinical history, ophthalmological examination and investigations. RESULTS: There were 66 new cases of IU, comprising 5.7% of 1168 new uveitis patients. The median age of diagnosis was 40 years (mean 39.4±15.9), with largest subgroup of the patients in the age group of 41-60 years (36.4%). The majority was Chinese (57.6%), followed by Asian Indians (18.2%) and Malays (16.7%). The ethnicity distribution was dissimilar to our ethnic distribution in Singapore (p<0.001) with an increased incidence of IU in the Asian Indian population. Most were idiopathic (59.1%) in etiology, followed by tuberculosis (TB) (15.2%). Ocular complications developed in 21 patients (31.8%), with cystoid macular edema (CME) being the commonest (28.8%). Severe vitritis occurred in 9.1% of patients, and was significantly associated with TB-associated IU (p<0.001). There was a downward trend for the incidence of the proportion of IU patients over the total uveitis patients (p = 0.021), with Spearman's rho of -0.786. CONCLUSIONS: Despite the downward trend, TB-associated IU was still of higher prevalence compared to less endemic areas, emphasizing the need for increased TB surveillance. A high index of suspicion for TB-associated IU is required in patients with severe vitritis. Comparisons with other countries revealed disparities in the IU etiologies, indicating possible geographical differences. Prevalence of known immune-mediated etiologies of IU is less compared to the western population. Our study also suggests a probable predisposition of the Singapore local Indian population for IU.

Comparative genetic analysis of VP4, VP1 and 3D gene regions of enterovirus 71 and coxsackievirus A16 circulating in Malaysia between 1997-2008.

Three genomic regions, VP4 capsid, VP1 capsid and 3D RNA polymerase of human enterovirus 71 (EV-71) and coxsackievirus A16 (CV-A16) were sequenced to understand the evolution of these viruses in Malaysia. A total of 42 EV-71 and 36 CV-A16 isolates from 1997- 2008 were sequenced. Despite the presence of many EV-71 subgenotypes worldwide, only subgenotypes B3, B4, B5, C1 and C2 were present in Malaysia. Importation of other subgenotypes such as C3, C4/D and C5 from other countries was infrequent. For CV-A16, the earlier subgenotype B1 was replaced by subgenotypes B2a and the recent B2c. Subgenotype B2a was present throughout the study while B2c only emerged in 2005. No genetic signatures could be attributed to viral virulence suggesting that host factors have a major role in determining the outcome of infection. Only three EV-71 B3 isolates showed non-consistent phylogeny in the 3D RNA polymerase region which indicated occurrence of recombination in EV-71. High genetic diversity was observed in the Malaysian EV-71 but Malaysian CV-A16 showed low genetic diversity in the three genomic regions sequenced. EV-71 showed strong purifying selection, but that occurred to a lesser extent in CV-A16.

Comparative genetic analysis of VP4, VP1 and 3D gene regions of enterovirus 71 and coxsackievirus A16 circulating in Malaysia between 1997-2008

Three genomic regions, VP4 capsid, VP1 capsid and 3D RNA polymerase of human enterovirus 71 (EV-71) and coxsackievirus A16 (CV-A16) were sequenced to understand the evolution of these viruses in Malaysia. A total of 42 EV-71 and 36 CV-A16 isolates from 1997-2008 were sequenced. Despite the presence of many EV-71 subgenotypes worldwide, only subgenotypes B3, B4, B5, C1 and C2 were present in Malaysia. Importation of other subgenotypes such as C3, C4/D and C5 from other countries was infrequent. For CV-A16, the earlier subgenotype B1 was replaced by subgenotypes B2a and the recent B2c. Subgenotype B2a was present throughout the study while B2c only emerged in 2005. No genetic signatures could be attributed to viral virulence suggesting that host factors have a major role in determining the outcome of infection. Only three EV-71 B3 isolates showed non-consistent phylogeny in the 3D RNA polymerase region which indicated occurrence of recombination in EV-71. High genetic diversity was observed in the Malaysian EV-71 but Malaysian CV-A16 showed low genetic diversity in the three genomic regions sequenced. EV-71 showed strong purifying selection, but that occurred to a lesser extent in CV-A16.

A porcine model of selective retinal capillary closure induced by embolization with fluorescent microspheres.

PURPOSE: To investigate the feasibility of creating an animal model of selective retinal capillary closure to mimic the capillary closure that occurs in diabetic retinopathy. METHODS: Fluorescent microspheres of 10- or 15-µm diameter were delivered to one eye of anesthetized pigs via a customized cannula advanced through the carotid arterial system to the origin of the external ophthalmic artery that supplies blood to the eye in this species. After preliminary trials in 10 pigs, embolization was performed in one eye of 34 animals that were allowed to survive for 7, 14, or 28 days. Embolized eyes were assessed by fluorescein angiography, electroretinography (ERG), and, after enucleation, light (LM) and electron (EM) microscopy. RESULTS: The microspheres were detectable in the retina immediately after embolization, were restricted to the nerve fiber layer of the retina, and remained thereafter within the retina for periods up to 28 days. They effectively occluded embolized capillaries and some precapillary arterioles. No systemic or cerebral adverse effects were noted, thus allowing survival and subsequent follow-up. Embolization caused a reduction in the b-wave amplitude and the oscillatory potentials of the rod-cone bright-flash ERG but did not affect the amplitude of the a-wave. Embolization induced extracellular and intracellular edema confined to the inner and mid retina, and as a result the retinas of embolized eyes were thicker than those of fellow, nonembolized eyes. The outer retina and RPE were unaffected. CONCLUSIONS: This survival model of retinal embolization with microspheres should be useful in the study of the retinal effects of the capillary closure that may occur in diabetic eyes.

Abdominal crisis in a young man with systemic lupus erythematosus.

Medium-sized artery aneurysms are rare in patients with systemic lupus erythematosus (SLE). We report on a 21-year-old Chinese man with SLE and secondary antiphospholipid syndrome (APS) who presented with acute abdominal pain due to a ruptured right hepatic artery aneurysm. He was also found to have aneurysms of the left hepatic artery and splenic artery on autopsy. There have been only eight cases of hepatic artery aneurysm and one case of splenic artery aneurysm associated with SLE in the English literature. Abdominal aneurysm must be suspected in SLE patients presenting with acute abdominal pain, haemoperitoneum or occult bleeding.

Effects of cyclic temperature changes on water sorption and solubility of composite restoratives.

This study investigated the effects of cyclic temperature changes on the water sorption and solubility of four commercial composite resins (Silux Plus, Z100, Ariston pHc and Surefil). The methodology was based upon ISO 4049 procedures with modifications for specimen dimension and thermal-cycling. Eighteen disc specimens (10 +/- 1 mm diameter and 1 +/- 0.1 mm thick) were made for each composite and randomly divided into three groups. The specimens were stored in a desiccator maintained at 35 +/- 1 degrees C until a constant mass was achieved and treated as follows: Group 1--stored in distilled water at 356 degrees C for 178 hrs; Group 2--stored in distilled water at 35 degrees C for 173 hours and subjected to five hours of thermal-cycling with an upper temperature of 45 degrees C; and Group 3--stored in distilled water at 35 degrees C for 173 hours and subjected to five hours of thermal-cycling with an upper temperature of 60 degrees C. Mass after treatment was measured and specimens were re-conditioned to constant mass. The volume of the specimens was obtained and water sorption/solubility calculated. Data was analyzed using factorial ANOVA/Scheffe's post-hoc test at significance level 0.05. The effects of thermal-cycling on water sorption was material dependent. Thermal-cycling at an upper temperature of 60 degrees C significantly increased water sorption of Silux Plus. A significant increase in water sorption was also observed when Z100 was thermal-cycled at an upper temperature of 45 degrees C. The water sorption of Ariston pHc and Surefil was not affected by thermal-cycling. Thermal-cycling did not affect the solubility of all composites. For all treatment groups, Surefil had significantly lower water sorption than the other composites evaluated. The water sorption of Z100 and Surefil was significantly lower than Silux Plus and Ariston pHc.

Effects of cyclic temperature changes on hardness of composite restoratives.

The clinical durability of some composite restorative materials may be significantly affected by cyclic temperature changes. This study investigated the effects of cyclic temperature changes on surface hardness of four commercial composite resins (Silux, Z100, Ariston and Surefil). Eighteen specimens of each material were divided into three treatment groups comprising a control and two different thermal cycling regimes. Control specimens were stored in distilled water at 35 degrees C for 178 hours. Thermal cycled specimens were stored in distilled water at 35 degrees C for 173 hours and subjected to five hours (300 cycles) of a thermal cycling regime consisting of the cycle ABAC, where A and B represent the fixed temperatures of 35 degrees C (28 seconds) and 15 degrees C (two seconds) and C, depending on the treatment group, either 45 degrees C or 60 degrees C (two seconds). All specimens were subsequently subjected to hardness testing (KHN) using a digital microhardness tester (load = 500 gf; dwell time = 15 seconds). Results were analyzed using ANOVA/Scheffe's test (p<0.05). The effect of thermal cycling on hardness was material-dependent. While thermal cycling significantly increased the surface hardness of Z100 and Surefil, it significantly decreased the hardness of Ariston. The hardness of Silux was not significantly affected by cyclic temperature changes. For all treatment groups, Z100 was significantly harder than the other composite resins evaluated and Surefil was significantly harder than Silux and Ariston. For both thermal cycled groups, Silux was significantly harder than Ariston.

Sudden death due to granulomatous myocarditis: a case of sarcoidosis?

INTRODUCTION: We report a case of sudden death due to granulomatous myocarditis and propose that cardiac sarcoid could have been the underlying aetiology. This is the first case reported in Singapore. The differential diagnoses for granulomatous myocarditis including sarcoidosis and its cardiac manifestations as well as idiopathic giant cell myocarditis are discussed. CLINICAL PICTURE: A 53-year-old Indian woman died suddenly and autopsy revealed bilateral hilar adenopathy and myocardial infiltrates which proved to be granulomatous in nature. CONCLUSION: Sarcoidosis may not be a rarity here and it is important to recognise the different clinical manifestations.

Influence of thermal cycling on OCA wear of composite restoratives.

This study investigated the effects of thermal cycling on wear of four commercial composite resins (Silux, Z100, Ariston and Surefil). Specimens of each material were divided into three treatment groups comprising a control and two different thermal cycling regimes. Control specimens were stored in distilled water at 35 degrees C for 178 hours. Thermal cycled specimens were stored in distilled water at 35 degrees C for 173 hours and subjected to five hours (300 cycles) of a thermal cycling regime consisting of the cycle ABAC, where A and B represent the fixed temperatures of 35 degrees C (28 seconds) and 15 degrees C (two seconds) and C, depending on the treatment group, was either 45 degrees C or 60 degrees C (two seconds). All specimens were subsequently subjected to wear testing at 20 MPa contact stress against SS304 counterbodies with distilled water as the lubricant. Wear depth (microm; n=6) was measured using profilometry every 2,000 cycles up to 10,000 cycles. Results were analyzed using ANOVA/Scheffe's test (p<0.05). The effect of thermal cycling on wear was material-dependent. The wear of Silux and Z100 were not significantly affected by thermal cycling. Thermal cycling of Ariston at an upper temperature of 60 degrees C significantly decreased wear resistance. Thermal cycling affected only the early wear resistance of Surefil.

Immunomagnetic isolation and long-term culture of mouse type A spermatogonia.

In the mammalian testis, type A spermatogonia proliferate and differentiate into sperm under the tight control of both endocrine and paracrine factors. In order to study the complex process of spermatogenesis at the molecular level, an in vitro system must be devised in which type A spermatogonia can be cultured for a prolonged period of time. Therefore, cocultures including type A spermatogonia and Sertoli cells, which act as nurse cells to the developing germ cells, are desirable. We have developed a method for the specific isolation of type A spermatogonia using magnetic beads and antibodies that recognize the c-kit receptor or the homophilic adhesion molecule, Ep-CAM. Purified spermatogonia could survive for a period of 25 days when cocultivated on Sertoli cell monolayers. Moreover, we recently established Sertoli cell lines that produce growth factors that are essential for the maintenance of spermatogonia in a proliferative state. Some of these Sertoli cell lines are able to reorganize into tubular structures when cultivated on a layer of Matrigel as extracellular matrix. We show here that type A spermatogonia associate specifically with the Sertoli cell tubules, and are able to replicate their DNA in this environment. Thus, these in vitro culture systems could be used for the long-term culture of primary, nonimmortalized type A spermatogonia.