Photochemical regeneration of flavoenzymes - An Old Yellow Enzyme case-study.

Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.

[Interventional treatment of benign prostatic hyperplasia : Embolization of the testicular vein]. / Interventionelle Therapie der benignen Prostatahyperplasie : Embolisation der V. spermatica.

CLINICAL/METHODICAL ISSUE: Embolization of the testicular veins (Gat-Goren technique) is an interventional procedure for the treatment of benign prostatic hyperplasia (BPH). STANDARD RADIOLOGICAL METHODS: In addition to standard urological procedures, such as transurethral resection of the prostate (TUR-P), arterial embolization of the prostate is considered as the interventional radiological method of choice. METHODICAL INNOVATIONS: Embolization of the testicular veins is technically identical to the embolization of varicoceles in infertile men and represents a less invasive treatment. PERFORMANCE: Embolization of the testicular veins can be performed as a low-risk intervention with low side effects. Variants of the venous anatomy can make the procedure more difficult. In current studies a good reduction of symptoms could be achieved in intermediate-term follow-up. ACHIEVEMENTS: The medium-term results are promising but data on long-term results and comparisons with alternative treatments are missing. PRACTICAL RECOMMENDATIONS: Embolization of the testicular veins is a minimally invasive method for the treatment of BPH. The procedure can be performed in an outpatient setting. In the intermediate course up to 6 months after treatment, promising results were demonstrated in recent studies. Randomized studies, data on long-term results and comparisons to alternative methods (e. g. arterial prostatic embolization and surgical procedures) are missing.

[High-intensity focused ultrasound (HIFU) for tumor pain relief in inoperable pancreatic cancer : Evaluation with the pain sensation scale (SES)]. / Hochintensiver fokussierter Ultraschall (HIFU) zur Linderung tumorbedingter Schmerzen bei inoperablem Pankreaskarzinom : Evaluation anhand der Schmerzempfindungsskala (SES).

BACKGROUND: High-intensity focused ultrasound (HIFU) in combination with palliative standard therapy is an innovative and effective treatment option for pain reduction in patients with inoperable pancreatic cancer. OBJECTIVE: Evaluation of the effects of additive ultrasound (US)-guided HIFU treatment in inoperable pancreatic cancer on the sensory and affective pain perception using validated questionnaries. MATERIAL AND METHODS: In this study 20 patients with locally advanced inoperable pancreatic cancer and tumor-related pain were treated by US-guided HIFU (6 stage III, 12 stage IV according to UICC and 2 with local recurrence after surgery). Ablation was performed using the JC HIFU system (HAIFU, Chongqing, China) with an ultrasonic device for real-time imaging. Clinical assessment included evaluation of pain severity using validated questionnaires with particular attention to the pain sensation scale (SES) with its affective and sensory component and the numeric rating scale (NRS). RESULTS: The average pain reduction after HIFU was 2.87 points on the NRS scale and 57.3 % compared to the mean baseline score (n = 15, 75 %) in 19 of 20 treated patients. Four patients did not report pain relief, however, the previous opioid medication could be stopped (n = 2) or the analgesic dosage could be reduced (n = 2). No pain reduction was achieved in one patient. Furthermore, after HIFU emotional as well as sensory pain aspects were significantly reduced (before vs. 1 week after HIFU, p < 0.05 for all pain scales). CONCLUSION: US-guided HIFU can be used for effective and early pain relief and reduction of emotional and sensory pain sensation in patients with locally advanced pancreatic cancer.

Three-Dimensional Isotropic Fat-Suppressed Proton Density-Weighted MRI at 3 Tesla Using a T/R-Coil Can Replace Multiple Plane Two-Dimensional Sequences in Knee Imaging.

PURPOSE: To evaluate whether a 3 D proton density-weighted fat-suppressed sequence (PDwFS) of the knee is able to replace multiplanar 2D-PDwFS. MATERIALS AND METHODS: 52 patients (26 men, mean age: 41.9 ±â€Š14.5years) underwent magnetic resonance imaging (MRI) of the knee at 3.0 Tesla using a T/R-coil. The imaging protocol included 3 planes of 2D-PDwFS (acquisition time (AT): 6:40 min; voxel sizes: 0.40 - 0.63 × 0.44 - 0.89 × 3mm³) and a 3D-PDwFS (AT: 6:31 min; voxel size: 0.63 × 0.68 × 0.63mm³). Homogeneity of fat suppression (HFS), artifacts, and image sharpness (IS) were evaluated on a 5-point scale (5[excellent] - 1[non-diagnostic]). The sum served as a measure for the overall image quality (OIQ). Contrast ratios (CR) compared to popliteal muscle were calculated for the meniscus (MEN), anterior (ACL) and posterior cruciate ligaments (PCL). In 13 patients who underwent arthroscopic knee surgery, two radiologists evaluated the presence of meniscal, ligamental and cartilage lesions to estimate the sensitivity and specificity of lesion detection. RESULTS: The CR was higher in the ACL, PCL and MEN in 3D- PDwFS compared to 2D-PDwFS (p < 0.01 for ACL and PCL; p = 0.07 for MEN). Compared to 2 D images, the OIQ was rated higher in 3D-PDwFS images (p < 0.01) due to fewer artifacts and HFS despite the lower IS (p < 0.01). The sensitivity and specificity of lesion detection in 3D- and 2D-PDwFS were similar. CONCLUSION: Compared to standard multiplanar 2D-PDwFS knee imaging, isotropic high spatial resolution 3D-PDwFS of the knee at 3.0 T can be acquired with high image quality in a reasonable scan time. Multiplanar reformations in arbitrary planes may serve as an additional benefit of 3D-PDwFS. KEY POINTS: • 3D-PDwFS of the knee is acquired with high image quality• 3D-PDwFS can be achieved in only one measurement with a reasonable scan time• 3D-PDwFS with the advantage of multiplanar reformation may replace 2D-PD-weighted knee MRI Citation Format: • Homsi R, Gieseke J, Luetkens JA et al. Three-Dimensional Isotropic Fat-Suppressed Proton Density-Weighted MRI at 3 Tesla Using a T/R-Coil Can Replace Multiple Plane Two-Dimensional Sequences in Knee Imaging. Fortschr Röntgenstr 2016; 188: 949 - 956.

Clinical Use of High-Intensity Focused Ultrasound (HIFU) for Tumor and Pain Reduction in Advanced Pancreatic Cancer.

PURPOSE: Evaluation of ultrasound-guided high-intensity focused ultrasound (HIFU) used for the first time in Germany in patients with inoperable pancreatic cancer for reduction of tumor volume and relief of tumor-associated pain. MATERIALS AND METHODS: 15 patients with locally advanced inoperable pancreatic cancer and tumor-related pain symptoms were treated by HIFU (n = 6 UICC stage III, n = 9 UICC stage IV). 13 patients underwent simultaneous standard chemotherapy. Ablation was performed using the JC HIFU system (Chongqing, China HAIFU Company) with an ultrasonic device for real-time imaging. Imaging follow-up (US, CT, MRI) and clinical assessment using validated questionnaires (NRS, BPI) was performed before and up to 15 months after HIFU. RESULTS: Despite biliary or duodenal stents (4/15) and encasement of visceral vessels (15/15), HIFU treatment was performed successfully in all patients. Treatment time and sonication time were 111 min and 1103 s, respectively. The applied total energy was 386 768 J. After HIFU ablation, contrast-enhanced imaging showed devascularization of treated tumor regions with a significant average volume reduction of 63.8 % after 3 months. Considerable pain relief was achieved in 12 patients after HIFU (complete or partial pain reduction in 6 patients). CONCLUSION: US-guided HIFU with a suitable acoustic pathway can be used for local tumor control and relief of tumor-associated pain in patients with locally advanced pancreatic cancer. KEY POINTS: • US-guided HIFU allows an additive treatment of unresectable pancreatic cancer.• HIFU can be used for tumor volume reduction.• Using HIFU, a significant reduction of cancer-related pain was achieved.• HIFU provides clinical benefit in patients with pancreatic cancer. Citation Format: • Strunk HM, Henseler J, Rauch M et al. Clinical Use of High-Intensity Focused Ultrasound (HIFU) for Tumor and Pain Reduction in Advanced Pancreatic Cancer. Fortschr Röntgenstr 2016; 188: 662 - 670.

Persistence and reactivation of human adenoviruses in the gastrointestinal tract.

Reactivation of persistent human adenoviruses (HAdVs) is associated with high morbidity and mortality in paediatric haematopoietic stem cell transplant (HSCT) recipients. Although invasive HAdV infections mainly arise from the gastrointestinal (GI) tract, the specific sites of HAdV persistence are not well characterised. We prospectively screened biopsies from 143 non-HSCT paediatric patients undergoing GI endoscopy and monitored serial stool specimens from 148 paediatric HSCT recipients for the presence of HAdV by real-time PCR. Persistence of HAdV in the GI tract was identified in 31% of children, with the highest prevalence in the terminal ileum. In situ hybridisation and immunohistochemistry identified HAdV persistence in lymphoid cells of the lamina propria, whereas biopsies from five transplant recipients revealed high numbers of replicating HAdV in intestinal epithelial cells. The prevalence of HAdV species, the frequencies of persistence in the GI tract and reactivations post transplant indicated a correlation of intestinal HAdV shedding pre-transplant with high risk of invasive infection. HAdV persistence in the GI tract is a likely origin of infectious complications in immunocompromised children. Intestinal lymphocytes represent a reservoir for HAdV persistence and reactivation, whereas the intestinal epithelium is the main site of viral proliferation preceding dissemination. The findings have important implications for assessing the risk of life-threatening invasive HAdV infections.

Novel Non-invasive Treatment With High-intensity Focused Ultrasound (HIFU).

Ultrasound is not only used for diagnostic purposes but it also can be applied therapeutically so far that nowadays high-intensity focused ultrasound (HIFU) even represents a novel non-invasive treatment modality for various solid tumors. HIFU works by causing selectively deep tissue destruction of target lesions within the body without harming adjacent and overlying structures. In this article, we present an overview on both the mode of action and requirements for a HIFU treatment as well as on the safety and the current status of indications and possible applications with regard to benign and malignant gynecological diseases. Based on numerous studies and original articles, HIFU proved to be an effective and low-risk treatment option particularly for uterine fibroids and adenomyosis, but it also seems to be effective for breast fibroadenomas or even for breast cancer in special cases and other rare entities.

Treatment of benign prostatic hyperplasia by occlusion of the impaired urogenital venous system - first experience.

PURPOSE: To effect regression of benign prostatic hyperplasia (BPH), Gat et al. (Andrologia 2008) proposed to occlude incompetent spermatic veins to reduce increased hydrostatic pressure on the prostatic venous plexus and prevent reflux with androgen rich blood from the testicles. Our aim was to implement this treatment strategy in clinical practice and to report about first results. METHODS: Embolization of the spermatic veins was performed in 30 patients with BPH. In 16 patients, we obtained follow-up data from at least 6 months. The sonographic transabdominal prostatic volume, prostate-specific antigen (PSA) and peripheral total testosterone levels were determined before and 6 months after the intervention. Subjective symptomatology was assessed using standardized questionnaires (International Prostate Symptom Score [IPSS] and Quality of Life score [QoL]) before and 6 months after the procedure. RESULTS: The age of all treated patients was 46 - 77 years. The age of the 16 patients who received follow-up was 51 - 77 years. IPSS (median 18 [IQR 20.75 - 14.50] vs. 9 [IQR 11.00 - 7.25], p < 0.0001) and QoL score (4 [IQR 5 - 3] vs. 2 [IQR 3 - 1], p < 0.001) were significantly decreased 6 months after the intervention. The subjective improvement of symptoms did not correspond with prostatic volumes, which did not change significantly (54.31 ±â€Š30.90 vs. 50.50 ±â€Š29.26 ml, p = n. s.). 4/16 patients had a measurable post-void urine volume, which decreased in two patients 6 months after the procedure, remained unchanged in one patient, and was no longer detectable in one patient. 4 of the 11 had a sonographically detectable varicocele before the intervention, and one patient had a trabeculated bladder. Both the peripheral total testosterone levels (4.55 ±â€Š1.27 vs. 3.93 ±â€Š1.00 ng/ml; p = n. s.) and PSA levels (3.74 ±â€Š2.83 vs. 4.06 ±â€Š3.34 ng/ml; p = n. s.) showed no significant differences. CONCLUSION: Interventional occlusion of the spermatic veins in patients with BPH is a feasible outpatient procedure with a low complication rate. Intermediate results are satisfactory. Mid- and long-term results and pathophysiologic mechanisms need to be further elucidated.

Successful treatment of partial portal vein thrombosis (PVT) with low dose rivaroxaban.

In a 63-year-old cirrhotic patient, recanalisation of a partial portal vein thrombosis was achieved by a low dose of rivaroxaban (10 mg daily). After anticoagulant therapy was stopped, partial vein thrombosis recurred. Restarting rivaroxaban at a dose of 10 mg led to recanalisation. The patient did not suffer any complications; in particular no bleeding occurred during 8 months of treatment.

Prognostic impact of meningeal dissemination in primary CNS lymphoma (PCNSL): experience from the G-PCNSL-SG1 trial.

BACKGROUND: We evaluated the frequency and prognostic impact of meningeal dissemination (MD) in immunocompetent adult patients with primary central nervous system lymphoma treated in a randomized phase III trial. PATIENTS AND METHODS: MD was evaluated at study entry and defined by lymphoma proof in the meningeal compartment detected by at least one of the following methods: cerebrospinal fluid (CSF) cytomorphology, detection of clonal B cells by IgH PCR in CSF or contrast enhancement of the leptomeninges on magnetic resonance imaging (MRI). RESULTS: Data on MD were available in 415 patients, of those, MD was detected in 65 (15.7%): in 44/361 (12.2%) by CSF cytomorphology, in 16/152 (10.5%) by PCR and in 17/415 (4.1%) by MRI. Major patients' characteristics and therapy did not significantly differ between patients with MD (MD+) versus those without MD (MD-). There was a significant correlation of MD with CSF pleocytosis (>5/µl; P < 0.0001), but no correlation with CSF protein elevation (>45 mg/dl). Median progression-free survival was 6.7 months [95% confidence interval (CI) 0-14.5] in MD+ and 8.3 months (5.7-10.8) in MD- patients (P = 0.95); median overall survival was 21.5 months (95% CI 16.8-26.1) and 24.9 months (17.5-32.3), respectively (P = 0.98). CONCLUSION: MD was detected infrequently and had no impact on outcome in this trial.

The potential for probiotic manipulation of the gastrointestinal microbiome.

Multiple internal and external sites of the healthy human body are colonized by a diversity of symbiotic microbes. The microbial assemblages found in the intestine represent some of the most dense and diverse of these human-associated ecosystems. Unsurprisingly, the enteric microbiome, that is the totality of microbes, their combined genomes, and their interactions with the human body, has a profound impact on physiological aspects of mammalian function, not least, host immune response. Lack of early-life exposure to certain microbes, or shifts in the composition of the gastrointestinal microbiome have been linked to the development and progression of several intestinal and extra-intestinal diseases, including childhood asthma development and inflammatory bowel disease. Modulating microbial exposure through probiotic supplementation represents a long-held strategy towards ameliorating disease via intestinal microbial community restructuring. This field has experienced somewhat of a resurgence over the past few years, primarily due to the exponential increase in human microbiome studies and a growing appreciation of our dependence on resident microbiota to modulate human health. This review aims to review recent regulatory aspects related to probiotics in food. It also summarizes what is known to date with respect to human gastrointestinal microbiota - the niche which has been most extensively studied in the human system - and the evidence for probiotic supplementation as a viable therapeutic strategy for modulating this consortium.

Monitoring of adenovirus load in stool by real-time PCR permits early detection of impending invasive infection in patients after allogeneic stem cell transplantation.

Invasive adenovirus (AdV) infections are associated with high morbidity and mortality in allogeneic stem cell transplant recipients. We observed that molecular detection of the virus in stool specimens commonly precedes AdV viremia, suggesting that intestinal infections may represent a common source of virus dissemination. To address this notion, we have investigated 153 consecutive allogeneic transplantations in 138 pediatric patients by quantitative monitoring of AdV in stool specimens and peripheral blood by a pan-adenovirus real-time (RQ)-PCR approach. AdV was detectable in serial stool specimens in all cases of AdV viremia during the post-transplant course (P<0.0001). The incidence of AdV viremia in individuals with peak virus levels in stool specimens above 1 x 10E6 copies per gram (n=22) was 73% vs 0% in patients with AdV levels in stool specimens below this threshold (n=29; P<0.0001). Serial measurement of AdV levels in stool specimens by RQ-PCR permitted early diagnosis of impending invasive infection with a sensitivity and specificity of 100% (95% confidence interval (CI) 96-100%) and 83% (95% CI 67-92%), respectively. The median time span between detection of AdV loads in stool specimens above 1 x 10E6 copies per gram and first observation of viremia was 11 days (range 0-192). Quantitative monitoring of the AdV load in stool specimens therefore provides a rationale for early initiation of antiviral treatment with the aim of preventing progression to life-threatening invasive infection.

Tacrolimus causes a blockage of protein secretion which reinforces its immunosuppressive activity and also explains some of its toxic side-effects.

BACKGROUND: Tacrolimus (FK506) is a macrolide immunosuppressant drug from the calcineurin inhibitor family, widely used in solid organ and islet cell transplantation, but produces significant side-effects. OBJECTIVE: This study examined the effect of FK506 on interleukin-2 (IL-2) and insulin secretion, establishing a novel characteristic of this drug that could explain its diverse adverse effects, and developed an experimental model for the simultaneous analysis of mRNA expression and protein secretion affected by this drug. METHODS: The IL-2 levels when tacrolimus was administered were analysed by Western blot, immunocytochemistry and RT-PCR in a T lymphocyte cellular line (Jurkat) 24 h post-stimulation. The insulin levels when tacrolimus was administered were analysed 4 h after stimulation of glucose-induced insulin secretion in a pancreatic cellular line (MIN6). RESULTS: The previously published information describes tacrolimus as only capable of partially blocking IL-2 mRNA expression. In our hands, the cellular content of IL-2 is almost undetectable in stimulated Jurkat cells and can be detected in cellular extracts only when the secretory pathway is blocked by brefeldin A (BFA). BFA added 2 h after the beginning of stimulation was able to inhibit IL-2 secretion, without affecting IL-2 mRNA expression. Therefore BFA utilization allowed us to establish a model to analyze the effect on IL-2 secretion, separately from its expression. Tacrolimus added before stimulation inhibits only IL-2 synthesis, but blocks IL-2 protein secretion when added 2 h after stimulation. Similarly, tacrolimus is also capable of blocking the glucose-stimulated secretion of insulin by MIN6 cells. An increase of the intracellular content can be detected concomitantly with a decrease of the hormone measured in the culture medium. CONCLUSIONS: Results of this study indicate that tacrolimus possesses another important effect in addition to the inhibition of IL-2 gene transcription, namely the ability to act as a general inhibitor of the protein secretory pathway. These results strongly suggest that the diabetogenic effect of the immune suppressant FK506 could be caused by the blockade of insulin secretion. This novel effect also provides an explanation for other side-effects observed in immunosuppressive treatment.

Endofacial competitive inhibition of the glucose transporter 1 activity by gossypol.

Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K(i) value of 20 microM. Likewise, GLUT1 fluorescence was quenched approximately 80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.

Safety and feasibility of long-term temozolomide treatment in patients with high-grade glioma.

We surveyed neuro-oncologists regarding patients treated with temozolomide for at least 12 cycles or 12 months. Patients receiving first-line temozolomide for a median 13 cycles had a median progression-free survival (PFS) of 14 months. Patients with recurrent disease receiving a median 14 cycles had a median PFS of 15.5 months. A small percentage of patients experienced grade III to IV toxicity. These results suggest that long-term treatment with temozolomide is feasible and well tolerated.

Novel identification of peripheral dopaminergic D2 receptor in male germ cells.

Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT-PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre- and post-meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non-capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology.

The afterglow of GRB 050709 and the nature of the short-hard gamma-ray bursts.

The final chapter in the long-standing mystery of the gamma-ray bursts (GRBs) centres on the origin of the short-hard class of bursts, which are suspected on theoretical grounds to result from the coalescence of neutron-star or black-hole binary systems. Numerous searches for the afterglows of short-hard bursts have been made, galvanized by the revolution in our understanding of long-duration GRBs that followed the discovery in 1997 of their broadband (X-ray, optical and radio) afterglow emission. Here we present the discovery of the X-ray afterglow of a short-hard burst, GRB 050709, whose accurate position allows us to associate it unambiguously with a star-forming galaxy at redshift z = 0.160, and whose optical lightcurve definitively excludes a supernova association. Together with results from three other recent short-hard bursts, this suggests that short-hard bursts release much less energy than the long-duration GRBs. Models requiring young stellar populations, such as magnetars and collapsars, are ruled out, while coalescing degenerate binaries remain the most promising progenitor candidates.

Gluconeogenesis-linked glycogen metabolism is important in the achievement of in vitro capacitation of dog spermatozoa in a medium without glucose.

In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [(14)C]glycogen after the addition to l-CCM with [(14)C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.