The role of tissue adhesives and sealants in colorectal anastomotic healing-a scoping review.

PURPOSE: Anastomotic leakage (AL) after colorectal resection is a serious postoperative complication with grave consequences for patients. Despite several efforts to reduce its incidence, AL is still seen among 2-20% of colorectal cancer patients receiving an anastomosis. The use of tissue adhesives and sealants as an extra layer of protection around the anastomosis has shown promising results. We conducted a scoping review to provide an overview of the current knowledge on the effect of tissue adhesives and sealants on colorectal anastomosis healing, as well as their effect on the postoperative outcome. METHODS: The databases of PubMed, Embase, and Cochrane Library were systematically searched on 14/10/2022. Studies addressing the use of a tissue adhesive or tissue sealant applied around a colorectal anastomosis, with the goal to prevent AL or to decrease AL-related complications, were included. We presented an overview of the available studies and summarized their results narratively. RESULTS: Seven studies were included out of the 846 screened. All authors reported the rate of AL in their interventions group. Five of the studies found a decreased rate of AL compared to the control group. One study had no incidences of AL, while the last study had a seemingly low rate of AL but no comparison group. Information on secondary outcomes was sparingly reported, but the results hinted at a positive effect. CONCLUSION: Tissue adhesives and sealants might have a beneficial effect on colorectal anastomosis healing. The literature is sparse, and this review has shown the need for further clinical studies.

Adaptor protein-3 produces synaptic vesicles that release phasic dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high-frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a distinct population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Adaptor Protein-3 Produces Synaptic Vesicles that Release Phasic Dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a novel population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Substrate recognition and proton coupling by a bacterial member of solute carrier family 17.

The solute carrier 17 family transports diverse organic anions using two distinct modes of coupling to a source of energy. Transporters that package glutamate and nucleotide into secretory vesicles for regulated release by exocytosis are driven by membrane potential but subject to allosteric regulation by H+ and Cl-. Other solute carrier 17 members including the lysosomal sialic acid exporter couple the flux of organic anion to cotransport of H+. To begin to understand how similar proteins can perform such different functions, we have studied Escherichia coli DgoT, a H+/galactonate cotransporter. A recent structure of DgoT showed many residues contacting D-galactonate, and we now find that they do not tolerate even conservative substitutions. In contrast, the closely related lysosomal H+/sialic acid cotransporter Sialin tolerates similar mutations, consistent with its recognition of diverse substrates with relatively low affinity. We also find that despite coupling to H+, DgoT transports more rapidly but with lower apparent affinity at high pH. Indeed, membrane potential can drive uptake, indicating electrogenic transport and suggesting a H+:galactonate stoichiometry >1. Located in a polar pocket of the N-terminal helical bundle, Asp46 and Glu133 are each required for net flux by DgoT, but the E133Q mutant exhibits robust exchange activity and rescues exchange by D46N, suggesting that these two residues operate in series to translocate protons. E133Q also shifts the pH sensitivity of exchange by DgoT, supporting a central role for the highly conserved TM4 glutamate in H+ coupling by DgoT.

Implementation of a standardized surgical technique in robot-assisted restorative rectal cancer resection: a single center cohort study.

BACKGROUND: Despite increasing focus on the technical performance of total mesorectal excision over recent decades, anastomotic leakage (AL) continues to be a serious complication for many patients, even in the hands of experienced surgical teams. This study describes implementation of standardized surgical technique in an effort to reduce variability, decrease the risk of anastomotic leakage, and improve associated short-term outcomes for rectal cancer patients undergoing robot-assisted restorative rectal resection (RRR). METHODS: We evaluated all rectal cancer patients undergoing robot-assisted RRR at Aarhus University Hospital between 2017 and 2020. Six standardized surgical steps directed to improve anastomotic healing were mandatory for all RRR. Additional changes were made during the period with prohibition of systemic dexamethasone and limiting the use of endoscopic stapling devices. RESULTS: The use of the full standardization, including all six surgical steps, increased from 40.3% (95% CI, 0.28-0.54) to 86.2% (95% CI, 0.68-0.95). The incidence of AL decreased from 21.0% (95% CI, 0.12-0.33) to 6.9% (95% CI, 0.01-0.23). Length of hospital stay (LOS) decreased from 6 days (range 2-50) to 5 days (range 2-26). The rate of patients readmitted within 90 days decreased from 21.0% (95% CI, 0.12-0.33), to 6.9% (95% CI, 0.01-0.23). CONCLUSION: The full standardization was effectively implemented for rectal cancer patients undergoing robot-assisted RRR. The risk of AL, LOS and readmission decreased during the study period. A team focus on high-reliability and peri-operative complications can improve patient outcomes.

Diversity of function and mechanism in a family of organic anion transporters.

Originally identified as transporters for inorganic phosphate, solute carrier 17 (SLC17) family proteins subserve diverse physiological roles. The vesicular glutamate transporters (VGLUTs) package the principal excitatory neurotransmitter glutamate into synaptic vesicles (SVs). In contrast, the closely related sialic acid transporter sialin mediates the flux of sialic acid in the opposite direction, from lysosomes to the cytoplasm. The two proteins couple in different ways to the H+ electrochemical gradient driving force, and high-resolution structures of the Escherichia coli homolog d-galactonate transporter (DgoT) and more recently rat VGLUT2 now begin to suggest the mechanisms involved as well as the basis for substrate specificity.

The impact of multiple firings on the risk of anastomotic leakage after minimally invasive restorative rectal cancer resection and the impact of anastomotic leakage on long-term survival: a population-based study.

PURPOSE: The aim of this study was to evaluate the anastomotic leakage (AL) rate and predictors for AL following minimally invasive restorative rectal resection (RRR) among rectal cancer patients managed according to up-to-date standardized treatment. Furthermore, we explored the impact of symptomatic AL on long-term survival. METHODS: The study cohort was rectal cancer patients undergoing minimally invasive RRR in Central Denmark Region between 2013 and 2017. Data was retrieved from a prospective clinical quality database and supplemented with data from medical records. The AL rate was calculated as the proportion of patients who developed symptomatic AL within 30 days. Predictors for AL were identified through logistic regression. The impact of AL on long-term survival was analyzed using Kaplan-Meier methods and Cox regression. RESULTS: AL occurred in 15.1% of 604 patients. The AL rate for males was 20.1% (95% CI 16.3-24.3) and 5.0% (95% CI 2.4-9.0) for females. Odds ratio (OR) of AL in females vs. males was 0.25 (95% CI 0.12-0.51). The use of at least three firings when transecting the rectum was associated with OR of 2.71 (95% CI 1.17-6.26) for AL. The 5-year survival for patients with vs. those without AL was 76.1% (95%CI 65.1-84.0) and 83.6% (95%CI 79.8-86.7), corresponding to adjusted hazard ratio of 1.43 (95%CI 0.84-2.41). CONCLUSION: Symptomatic AL is still a challenge in a standardized setting using minimally invasive surgery in rectal cancer patients undergoing RRR, especially in men. Multiple firings should be avoided in transection of the rectum with an endoscopic stapler. AL had a statistical non-significant negative impact on survival.

Anastomotic leakage following restorative rectal cancer resection: treatment and impact on stoma presence 1 year after surgery-a population-based study.

PURPOSE: Anastomotic leakage (AL) continues to be a challenge after restorative rectal resection (RRR). Various treatment options of AL are available; however, their long-term outcomes are uncertain. We explored the impact of AL on the risk of stoma presence 1 year after RRR for rectal cancer and described treatment of AL after RRR including impact on the probability of receiving adjuvant chemotherapy and stoma presence following different treatment options of AL. METHODS: We included 859 patients undergoing RRR in Central Denmark Region between 2013 and 2019. Stoma presence was calculated as the proportion of patients with stoma 1 year after RRR. Multivariable logistic regression was conducted to estimate the impact of AL on stoma presence adjusting for potential predictors. Descriptive data of outcomes were stratified for various treatment options of AL. RESULTS: The risk of stoma presence 1 year after surgery was 9.8% (95% CI 7.98-12.0). Predictors for having stoma 1 year after RRR were AL (OR 8.43 (95% CI 4.87-14.59)) and low tumour height (OR 3.85 (95% CI 1.22-13.21)). For patients eligible for adjuvant chemotherapy, the probability of receiving it was 42.9% (95% CI 21.8-66.0) if treated with endo-SPONGE and 71.4% (95% CI 47.8-88.7) if treated with other anastomosis preserving treatment options. The risk of having stoma 1 year after RRR was 33.9% (95% CI 21.8-47.8) for patients treated with endo-SPONGE and 13.5% (95% CI 5.6-25.8) for patients treated with other anastomosis preserving treatment options (p = 0.013). CONCLUSION: AL is a strong predictor for stoma presence 1 year after RRR. Patients treated with endo-SPONGE seem to have worse outcomes compared to other anastomosis preserving treatment options.

Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

Ion transport and regulation in a synaptic vesicle glutamate transporter.

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo-electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.

The mechanism and regulation of vesicular glutamate transport: Coordination with the synaptic vesicle cycle.

The transport of classical neurotransmitters into synaptic vesicles generally relies on a H+ electrochemical gradient (∆µH+). Synaptic vesicle uptake of glutamate depends primarily on the electrical component ∆ψ as the driving force, rather than the chemical component ∆pH. However, the vesicular glutamate transporters (VGLUTs) belong to the solute carrier 17 (SLC17) family, which includes closely related members that function as H+ cotransporters. Recent work has also shown that the VGLUTs undergo allosteric regulation by H+ and Cl-, and exhibit an associated Cl- conductance. These properties appear to coordinate VGLUT activity with the large ionic shifts that accompany the rapid recycling of synaptic vesicles driven by neural activity. Recent structural information also suggests common mechanisms that underlie the apparently divergent function of SLC17 family members, and that confer allosteric regulation.

Resting state structure of the hyperdepolarization activated two-pore channel 3.

Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated sodium channel, two-pore channel 3 (TPC3), which generates ultralong action potentials. TPC3 is distinguished by activation only at extreme membrane depolarization (V50 ∼ +75 mV), in contrast to other TPCs and NaV channels that activate between -20 and 0 mV. We present electrophysiological evidence that TPC3 voltage activation depends only on voltage sensing domain 2 (VSD2) and that each of the three gating arginines in VSD2 reduces the activation threshold. The structure presents a chemical basis for sodium selectivity, and a constricted gate suggests a closed pore consistent with extreme voltage dependence. The structure, confirmed by our electrophysiology, illustrates the configuration of a bona fide resting state voltage sensor, observed without the need for any inhibitory ligand, and independent of any chemical or mutagenic alteration.

Diagnostic accuracy of ultrasound-guided core biopsy of peripheral nerve sheath tumors.

PURPOSE: To retrospectively evaluate the diagnostic accuracy of and complications from ultrasound-guided core needle biopsy (UGCNB) of suspected peripheral nerve sheath tumors (PNSTs). METHODS: Patients undergoing UGCNB from January 2004 to December 2016, based on the suspicion of PNST, were included in the study. Age, gender, anatomical location, dates of relevant events, and histopathological reports of the UGCNB cores and the resected tumors were retrieved from the patients' medical records. RESULTS: A total of 154 UGCNBs were identified. One hundred and forty (90.9%) of these resulted in a conclusive histopathological report, while 14 were unsuited for histopathological analysis due to insufficient amount of tissue and/or nonrepresentative tissue. The overall diagnostic accuracy of UGCNB with respect to discriminate malignant from benign tumors was 99.3%, while correct specific UGCNB diagnoses were confirmed in 95.1% of the cases. Sensitivity and specificity were 90.9% (95% CI: 58.7-99.8%) and 100% (95% CI: 97.2-100%), respectively. The positive predictive value was 100%, and the negative predictive value was 99.2%. Except for one patient, who reported mild dysesthesia, which resolved 2 days after the UGCNB, no complications were reported. CONCLUSION: This study suggests that UGCNB is accurate and safe in patients suspected for PNST.

Structures suggest a mechanism for energy coupling by a family of organic anion transporters.

Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane.

Synaptic Vesicle Recycling Pathway Determines Neurotransmitter Content and Release Properties.

In contrast to temporal coding by synaptically acting neurotransmitters such as glutamate, neuromodulators such as monoamines signal changes in firing rate. The two modes of signaling have been thought to reflect differences in release by different cells. We now find that midbrain dopamine neurons release glutamate and dopamine with different properties that reflect storage in different synaptic vesicles. The vesicles differ in release probability, coupling to presynaptic Ca2+ channels and frequency dependence. Although previous work has attributed variation in these properties to differences in location or cytoskeletal association of synaptic vesicles, the release of different transmitters shows that intrinsic differences in vesicle identity drive different modes of release. Indeed, dopamine but not glutamate vesicles depend on the adaptor protein AP-3, revealing an unrecognized linkage between the pathway of synaptic vesicle recycling and the properties of exocytosis. Storage of the two transmitters in different vesicles enables the transmission of distinct signals.

The dual role of chloride in synaptic vesicle glutamate transport.

The transport of glutamate into synaptic vesicles exhibits an unusual form of regulation by Cl- as well as an associated Cl- conductance. To distinguish direct effects of Cl- on the transporter from indirect effects via the driving force Δψ, we used whole endosome recording and report the first currents due to glutamate flux by the vesicular glutamate transporters (VGLUTs). Chloride allosterically activates the VGLUTs from both sides of the membrane, and we find that neutralization of an arginine in transmembrane domain four suffices for the lumenal activation. The dose dependence suggests that Cl- permeates through a channel and glutamate through a transporter. Competition between the anions nonetheless indicates that they use a similar permeation pathway. By controlling both ionic gradients and Δψ, endosome recording isolates different steps in the process of synaptic vesicle filling, suggesting distinct roles for Cl- in both allosteric activation and permeation.

Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains.

Dopamine regulates reward, cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. Here, we use super-resolution imaging to show that the dopamine transporter is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to, but not overlapping with, the dopamine transporter nanodomains. The molecular organization of the dopamine transporter in nanodomains is reversibly reduced by short-term activation of NMDA-type ionotropic glutamate receptors, implicating dopamine transporter nanodomain distribution as a potential mechanism to modulate dopaminergic neurotransmission in response to excitatory input.The dopamine transporter (DAT) has a crucial role in the regulation of neurotransmission. Here, the authors use super-resolution imaging to show that DAT clusters into cholesterol-dependent membrane regions that are reversibly regulated by ionotropic glutamate receptors activation.

Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism.

The quantal nature of synaptic transmission requires a mechanism to transport neurotransmitter into synaptic vesicles without promoting non-vesicular efflux across the plasma membrane. Indeed, the vesicular transport of most classical transmitters involves a mechanism of H(+) exchange, which restricts flux to acidic membranes such as synaptic vesicles. However, vesicular transport of the principal excitatory transmitter glutamate depends primarily on membrane potential, which would drive non-vesicular efflux, and the role of protons is unclear. Adapting electrophysiology to record currents associated with the vesicular glutamate transporters (VGLUTs), we characterize a chloride conductance that is gated by lumenal protons and chloride and supports glutamate uptake. Rather than coupling stoichiometrically to glutamate flux, lumenal protons and chloride allosterically activate vesicular glutamate transport. Gating by protons serves to inhibit what would otherwise be substantial non-vesicular glutamate efflux at the plasma membrane, thereby restricting VGLUT activity to synaptic vesicles.

A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity.

Midbrain dopaminergic (DAergic) neurons are a heterogeneous cell group, composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting only 20 000-30 000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, a bacterial artificial chromosome mouse line [Dat1-enhanced green fluorescent protein (eGFP)] from the Gene Expression Nervous System Atlas (GENSAT) that expresses eGFP under control of the dopamine transporter (DAT) promoter was characterized. Confocal microscopy analysis of brain sections showed strong eGFP signal reporter in midbrain regions and striatal terminals that co-localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co-localization of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP-expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra and ventral tegmental area. Dat1-eGFP mice showed neither change in synaptosomal DA uptake nor altered levels of DAT and TH in both striatum and midbrain. No behavioural difference between Dat1-eGFP and wild-type was found, suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence-activated cell sorting and the sorted neurons can be cultured in vitro. The current investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1-eGFP mouse strain constitutes a promising tool for delineating new aspects of DA biology.

The serotonin transporter undergoes constitutive internalization and is primarily sorted to late endosomes and lysosomal degradation.

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the "long loop" recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the ß2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation.