High-resolution macromolecular crystallography at the FemtoMAX beamline with time-over-threshold photon detection.

Protein dynamics contribute to protein function on different time scales. Ultrafast X-ray diffraction snapshots can visualize the location and amplitude of atom displacements after perturbation. Since amplitudes of ultrafast motions are small, high-quality X-ray diffraction data is necessary for detection. Diffraction from bovine trypsin crystals using single femtosecond X-ray pulses was recorded at FemtoMAX, which is a versatile beamline of the MAX IV synchrotron. The time-over-threshold detection made it possible that single photons are distinguishable even under short-pulse low-repetition-rate conditions. The diffraction data quality from FemtoMAX beamline enables atomic resolution investigation of protein structures. This evaluation is based on the shape of the Wilson plot, cumulative intensity distribution compared with theoretical distribution, I/σ, Rmerge/Rmeas and CC1/2 statistics versus resolution. The FemtoMAX beamline provides an interesting alternative to X-ray free-electron lasers when studying reversible processes in protein crystals.

Accurate population-based model for individual prediction of colon cancer recurrence.

BACKGROUND: Prediction models are useful tools in the clinical management of colon cancer patients, particularly when estimating the recurrence rate and, thus, the need for adjuvant treatment. However, the most used models (MSKCC, ACCENT) are based on several decades-old patient series from clinical trials, likely overestimating the current risk of recurrence, especially in low-risk groups, as outcomes have improved over time. The aim was to develop and validate an updated model for the prediction of recurrence within 5 years after surgery using routinely collected clinicopathologic variables. MATERIAL AND METHODS: A population-based cohort from the Swedish Colorectal Cancer Registry of 16,134 stage I-III colon cancer cases was used. A multivariable model was constructed using Cox proportional hazards regression. Three-quarters of the cases were used for model development and one quarter for internal validation. External validation was performed using 12,769 stage II-III patients from the Norwegian Colorectal Cancer Registry. The model was compared to previous nomograms. RESULTS: The nomogram consisted of eight variables: sex, sidedness, pT-substages, number of positive and found lymph nodes, emergency surgery, lymphovascular and perineural invasion. The area under the curve (AUC) was 0.78 in the model, 0.76 in internal validation, and 0.70 in external validation. The model calibrated well, especially in low-risk patients, and performed better than existing nomograms in the Swedish registry data. The new nomogram's AUC was equal to that of the MSKCC but the calibration was better. CONCLUSION: The nomogram based on recently operated patients from a population registry predicts recurrence risk more accurately than previous nomograms. It performs best in the low-risk groups where the risk-benefit ratio of adjuvant treatment is debatable and the need for an accurate prediction model is the largest.

Role of Thermal Equilibrium Dynamics in Atomic Motion during Nonthermal Laser-Induced Melting.

This study shows that initial atomic velocities as given by thermodynamics play an important role in the dynamics of phase transitions. We tracked the atomic motion during nonthermal laser-induced melting of InSb at different initial temperatures. The ultrafast atomic motion following bond breaking can in general be governed by two mechanisms: the random velocity of each atom at the time of bond breaking (inertial model), and the forces acting on the atoms after bond breaking. The melting dynamics was found to follow the inertial model over a wide temperature range.

FemtoMAX - an X-ray beamline for structural dynamics at the short-pulse facility of MAX IV.

The FemtoMAX beamline facilitates studies of the structural dynamics of materials. Such studies are of fundamental importance for key scientific problems related to programming materials using light, enabling new storage media and new manufacturing techniques, obtaining sustainable energy by mimicking photosynthesis, and gleaning insights into chemical and biological functional dynamics. The FemtoMAX beamline utilizes the MAX IV linear accelerator as an electron source. The photon bursts have a pulse length of 100 fs, which is on the timescale of molecular vibrations, and have wavelengths matching interatomic distances (Å). The uniqueness of the beamline has called for special beamline components. This paper presents the beamline design including ultrasensitive X-ray beam-position monitors based on thin Ce:YAG screens, efficient harmonic separators and novel timing tools.

Salivary secretion in health and disease.

Saliva is a complex fluid produced by 3 pairs of major salivary glands and by hundreds of minor salivary glands. It comprises a large variety of constituents and physicochemical properties, which are important for the maintenance of oral health. Saliva not only protects the teeth and the oropharyngeal mucosa, it also facilitates articulation of speech, and is imperative for mastication and swallowing. Furthermore, saliva plays an important role in maintaining a balanced microbiota. Thus, the multiple functions provided by saliva are essential for proper protection and functioning of the body as a whole and for the general health. A large number of diseases and medications can affect salivary secretion through different mechanisms, leading to salivary gland dysfunction and associated oral problems, including xerostomia, dental caries and fungal infections. The first part of this review article provides an updated insight into our understanding of salivary gland structure, the neural regulation of salivary gland secretion, the mechanisms underlying the formation of saliva, the various functions of saliva and factors that influence salivary secretion under normal physiological conditions. The second part focuses on how various diseases and medical treatment including commonly prescribed medications and cancer therapies can affect salivary gland structure and function. We also provide a brief insight into how to diagnose salivary gland dysfunction.

Electrostimulation of the lingual nerve by an intraoral device may lead to salivary gland regeneration: A case series study.

BACKGROUND: Salivary gland function is controlled by the salivary reflex, whose efferent arm is composed by the parasympathetic and the sympathetic divisions of the autonomic nervous system. Parenchymal injury is the main salivary gland involvement of Sjögren's syndrome and head and neck radiotherapy, but neural damage has been reported as well. Recently an intraoral device for electrostimulation of the lingual nerve in vicinity to the lower third molar has been introduced. At this point this nerve carries efferent fibers for the innervation of the submandibular, sublingual and several minor salivary glands and afferent fibers of the salivary reflex. Therefore, excitation of these fibers potentially leads to increased secretion of all salivary glands. Thus, the study objective was to assess whether comprehensive neural activation by electrostimulation of the lingual nerve carries the potential to induce the regeneration of damaged salivary glands. MATERIAL AND METHODS: The device was tested on three patients with no collectable resting and stimulated secretion of saliva during a double blind, sham controlled period of two months and nine open-label months. RESULTS: All three subjects developed the capacity to spit saliva, not only in direct response to the electrostimulation but also after free intervals without electrostimulation. In addition, their symptoms of dry mouth severity and frequency improved. CONCLUSIONS: This recovery is probably due to the combined effect of increase in secretory functional gland mass and regain of nervous control of the secretory elements and blood vessels. Both are phenomena that would contribute to gland regeneration.

[Medicaments and oral healthcare. Systematic review of the -literature assessing the effect of drugs on the salivary glands]. / Serie: Medicamenten en mondzorg.Systematisch literatuuronderzoek naar effect van medicatie op de speekselklieren.

Evidence-based reviews of drugs causing medication-induced salivary gland dysfunction, such as xerostomia (sensation of oral dryness) and subjective sialorrhea are lacking. To compile a list of medicaments that influence salivary gland function, electronic databases were searched for relevant articles published up to June 2013. A total of 269 papers out of 3,867 records located satisfied the inclusion criteria (relevance, quality of methodology, strength of evidence). A total of 56 active substances with a higher level of evidence and 50 active substances with a moderate level of evidence of causing salivary gland dysfunction are described in this article. While xerostomia was a commonly reported outcome, the objective effect on salivary secretion was rarely measured. Xerostomia was, moreover, mostly reported as a negative side effect instead of the intended effect of that drug. A comprehensive list of medications having documented effects on salivary gland function or symptoms was compiled, which may assist practitioners in assessing patients who complain of dry mouth while taking medications.

World Workshop on Oral Medicine VI: a systematic review of medication-induced salivary gland dysfunction.

The aim of this paper was to perform a systematic review of the pathogenesis of medication-induced salivary gland dysfunction (MISGD). Review of the identified papers was based on the standards regarding the methodology for systematic reviews set forth by the World Workshop on Oral Medicine IV and the PRISMA statement. Eligible papers were assessed for both the degree and strength of relevance to the pathogenesis of MISGD as well as on the appropriateness of the study design and sample size. A total of 99 papers were retained for the final analysis. MISGD in human studies was generally reported as xerostomia (the sensation of oral dryness) without measurements of salivary secretion rate. Medications may act on the central nervous system (CNS) and/or at the neuroglandular junction on muscarinic, α-and ß-adrenergic receptors and certain peptidergic receptors. The types of medications that were most commonly implicated for inducing salivary gland dysfunction were those acting on the nervous, cardiovascular, genitourinary, musculoskeletal, respiratory, and alimentary systems. Although many medications may affect the salivary flow rate and composition, most of the studies considered only xerostomia. Thus, further human studies are necessary to improve our understanding of the association between MISGD and the underlying pathophysiology.

Effect of the neuropeptides vasoactive intestinal peptide, peptide histidine methionine and substance P on human major salivary gland secretion.

OBJECTIVE: The parasympathetic transmitters vasoactive intestinal peptide (VIP) and substance P (SP) are secretagogues in salivary glands of animals. Currently, we hypothesise that in human salivary glands, these neuropeptides and the VIP-related peptide histidine methionine (PHM) also exert secretory actions, reflected morphologically by exocytosis of acinar protein/glycoprotein-storing granules. MATERIALS AND METHODS: Submandibular and parotid gland tissues, exposed in vitro to VIP and PHM, and SP, respectively, were examined by light and transmission electron microscopy. For comparison, the response to in vitro stimulation of isoproterenol, phenylephrine and carbachol was examined. Moreover, the peptidergic innervation of the glands was examined by immunohistochemistry. RESULTS: Vasoactive intestinal peptide- and PHM-immunoreactive nerves were in close proximity to acini and ducts in the two glands, while these elements lacked a SP-positive innervation. While no morphological changes occurred in response to SP (parotid glands), VIP and PHM administration (submandibular glands) caused conspicuous acinar degranulation accompanied by luminal space broadening. In the two glands, both α1 - and ß-adrenergic receptor stimulation and muscarinic receptor stimulation caused similar changes as to VIP/PHM, although to varying extent. CONCLUSIONS: Vasoactive intestinal peptide and PHM, but not SP, are likely transmitters in the parasympathetic control of salivary (protein) secretion in humans.

Improved replication efficiency of echovirus 5 after transfection of colon cancer cells using an authentic 5' RNA genome end methodology.

Oncolytic virotherapy is a promising novel form of cancer treatment, but the therapeutic efficiency needs improvement. A potential strategy to enhance the therapeutic effect of oncolytic viruses is to use infectious nucleic acid as therapeutic agent to initiate an oncolytic infection, without administrating infectious viral particles. Here we demonstrate improved viral replication activation efficiency when transfecting cells with 5' end authentic in vitro transcribed enterovirus RNA as compared to genomic RNA with additional non-genomic 5' nucleotides generated by conventional cloning methods. We used echovirus 5 (E5) as an oncolytoc model virus due to its ability to replicate in and completely destroy five out of six colon cancer cell lines and kill artificial colon cancer tumors (HT29 spheroids), as shown here. An E5 infectious cDNA clone including a hammerhead ribozyme sequence was used to generate in vitro transcripts with native 5' genome ends. In HT29 cells, activation of virus replication is approximately 20-fold more efficient for virus genome transcripts with native 5' genome ends compared to E5 transcripts generated from a standard cDNA clone. This replication advantage remains when viral progeny release starts by cellular lysis 22 h post transfection. Hence, a native 5' genomic end improves infection activation efficacy of infectious nucleic acid, potentially enhancing its therapeutic effect when used for cancer treatment. The clone design with a hammerhead ribozyme is likely to be applicable to a variety of oncolytic positive sense RNA viruses for the purpose of improving the efficacy of oncolytic virotherapy.

Parasympathetic vasoactive intestinal peptide (VIP): a likely contributor to clozapine-induced sialorrhoea.

OBJECTIVE: The parasympathetic transmitter vasoactive intestinal peptide (VIP) increases salivary gland blood flow and evokes protein secretion and, in some species, such as rats, a small fluid secretion. It interacts synergistically with muscarinics for protein and fluid output. Human salivary acini are supplied with VIP-containing nerves. We hypothesise that VIP and clozapine, acting together, evoke a volume of saliva greater than the sum of those induced by each drug given separately. It was further considered whether, in the current test situation, circulatory events influenced the magnitude of the secretory response. MATERIAL AND METHODS: Saliva from parotid glands deprived of their autonomic innervation, and saliva and blood from innervated submandibular glands were collected in adrenoceptor antagonist-pretreated pentobarbitone-anaesthetised rats. Initially, the individual and then the combined effects of intravenous doses of VIP and clozapine were established. RESULTS: The submandibular volume response to the combination was 2-3 times higher, while blood pressure and glandular blood flow did not differ from those to VIP alone. The synergism occurred independent of nerves as shown in denervated parotid glands. CONCLUSIONS: From the current preclinical data, we speculate that VIP of parasympathetic origin, by its synergistic interaction with clozapine, may contribute to the clozapine (muscarinic M1-receptor)-induced sialorrhoea in schizophrenics.

The antipsychotic amisulpride: ultrastructural evidence of its secretory activity in salivary glands.

OBJECTIVE: Amisulpride is reported to inhibit clozapine-induced sialorrhea. Preclinically, clozapine evokes muscarinic-M1-type-mediated secretion that, however, amisulpride does not reduce. Instead, amisulpride, without causing any overt secretion per se, enhances both nerve- and autonomimetic-evoked salivation by unknown mechanism(s). Hypothesizing that amisulpride prepares the gland for secretion, we looked for ultrastructural events indicating secretory activity in intercellular canaliculi of serous/seromucous cells, that is, density increase in protrusions (reflecting anchored granules) and in microbuds (reflecting recycling membranes and/or vesicle secretion) and decrease in microvilli (reflecting the cytoskeletal re-arrangement related to exocytosis). MATERIAL AND METHODS: Rat parotid and submandibular glands were exposed to amisulpride in vivo or in vitro. Glands were processed for transmission electron and scanning electron microscopy and then morphometrically assessed. RESULTS: Cells were packed with secretory granules. The density of protrusions increased in both glands, whereas significant and parallel changes in microvilli and microbuds occurred only in parotid glands, and in vitro. CONCLUSIONS: Amisulpride induced ultrastructural signs of secretory activity but to varying extent; in submandibular glands, in contrast to parotid glands, changes were not brought beyond the granular anchoring stage. Amisulpride may provide an overall readiness for secretion that will result in augmented responses to agonists, a phenomenon of potential interest in dry-mouth treatment.

Subcellular distribution of melatonin receptors in human parotid glands.

The hormone melatonin influences oral health through a variety of actions, such as anti-inflammatory, anti-oxidant, immunomodulatory and antitumour. Many of these melatonin functions are mediated by a family of membrane receptors expressed in the oral epithelium and salivary glands. Using immunoblotting and immunohistochemistry, recent studies have shown that the melatonin membrane receptors, MT1 and MT2, are present in rat and human salivary glands. To date, no investigation has dealt with the ultrastructural distribution of the melatonin receptors. This was the aim of the present study, using the immunogold method applied to the human parotid gland. Reactivity to MT1 and, with less intensity, to MT2 appeared in the secretory granules of acinar cells and in the cytoplasmic vesicles of both acinar and ductal cells. Plasma membranes were also stained, albeit slightly. The peculiar intracytoplasmic distribution of these receptors may indicate that there is an uptake/transport system for melatonin from the circulation into the saliva.

Salivary secretion effects of the antipsychotic drug olanzapine in an animal model.

OBJECTIVE: Olanzapine, introduced as an alternative to clozapine in schizophrenia therapy, is thought to display a receptor affinity similar to that of clozapine. Antipsychotics are well-known xerogenic drugs. However, clozapine exerts both antagonistic and agonistic salivary effects ('clozapine-induced sialorrhea'), the latter probably via muscarinic M1 type of receptor. We hypothesise that olanzapine also has dual salivary effects. MATERIAL AND METHODS: Effects of intravenous olanzapine were examined in rats, including those subjected to chronic preganglionic parasympathetic denervation (submandibular glands) or combined postganglionic parasympathetic and sympathetic denervation (parotid glands). Secretion was evoked reflexly, and by intravenous methacholine and the tachykinin substance P. RESULTS: At 0.01-1 mg kg(-1), olanzapine dose dependently reduced secretion in response to methacholine or reflex stimulus but not that to substance P. At 10 mg kg(-1), olanzapine evoked a long-lasting secretion, independent of the autonomic innervation as well as of α- and ß-adrenergic receptors and muscarinic receptors. The secretion was reduced, but not abolished, by a substance P receptor antagonist. CONCLUSIONS: Like clozapine, olanzapine evoked secretion. The response to olanzapine was greater and, in contrast to clozapine, involved non-traditional gland receptors (such as substance P receptors). The findings imply that olanzapine plays an excitatory role via tachykinin receptors in humans.

No evidence of enteroviruses in the intestine of patients with type 1 diabetes.

AIMS/HYPOTHESIS: The purpose of this study was to investigate whether the gut mucosa is a reservoir for enterovirus persistence in patients with type 1 diabetes. METHODS: Small intestine biopsy samples from 25 individuals at different stages of type 1 diabetes, 21 control individuals and 27 individuals with coeliac disease were analysed for the presence of enterovirus RNA by using both radioactive in-situ hybridisation and real-time RT-PCR and for the presence of enterovirus proteins by immunostaining with antibodies against VP1 and VP4-2-3 capsid proteins and virus polymerase. Lymphocytic enteropathy and serum anti-VP1 antibodies were also evaluated at the time of biopsy. Moreover, high-throughput sequencing was performed to identify viral transcripts or genomes. RESULTS: Enterovirus was not detected by in-situ hybridisation or RT-PCR in any of the individuals tested. Immunohistology revealed a few stained cells in the intestinal epithelium in a low number of individuals, with no difference between diabetic and non-diabetic individuals. Levels of serum IgG against VP1 did not differ between control individuals and those with diabetes or coeliac disease and no evidence of diabetes-related lymphocytic enteropathy was detected. High-throughput sequencing did not reveal specific enterovirus sequences in the gut mucosa of individuals with type 1 diabetes. CONCLUSIONS/INTERPRETATION: Prolonged/persistent enterovirus infections in gut mucosa are not common in patients with type 1 diabetes.

Atypical antipsychotics--effects of amisulpride on salivary secretion and on clozapine-induced sialorrhea.

OBJECTIVE: Amisulpride is suggested for treatment of clozapine-induced sialorrhea. However, objective measurements of its effectiveness are lacking and, preclinically, amisulpride has no effect. We currently hypothesise that amisulpride acts by reducing the nervous- rather than the clozapine-driven salivary secretion. MATERIAL AND METHODS: Effects of intravenous amisulpride (as well as of clozapine and raclopride, a dopamine D2/D3 antagonist) were investigated in rats, including those subjected to chronic preganglionic parasympathetic denervation (submandibular glands) or combined postganglionic parasympathetic and sympathetic denervation (parotid glands). In duct-cannulated glands, secretion was evoked reflexly, at low and maximum flow rates, and by electrical stimulation of the parasympathetic and sympathetic innervations, and administration of autonomimetics (including substance P). RESULTS: Unlike clozapine, amisulpride had no effect on the reflexly evoked secretion at maximum rate. With respect to reflex secretion at low rate and to the secretion evoked by muscarinic, α-adrenergic, ß-adrenergic and substance P receptors, amisulpride (in contrast to raclopride) dose dependently potentiated the responses. Amisulpride had no effect on gland blood flow. CONCLUSIONS: No support for any inhibitory influence of amisulpride was found. Conversely, amisulpride universally enhanced secretion, suggesting that amisulpride is a potential drug for dry-mouth treatment. The mechanism behind the potentiation is currently unknown.

Anti-inflammatory action of cholecystokinin and melatonin in the rat parotid gland.

OBJECTIVE: To define the influence of cholecystokinin and melatonin on the inflammatory response of the lipopolysaccharide-exposed rat parotid gland. MATERIALS AND METHODS: Bacterial lipopolysaccharide was infused retrogradely into the parotid duct. The degree of inflammation three hours postadministration was estimated from the activity of myeloperoxidase, reflecting glandular neutrophil infiltration. RESULTS: The myeloperoxidase activity of the lipopolysaccharide-exposed gland was 10-fold greater than that of the contralateral gland. Combined with sulphated cholecystokinin-8 (10 or 25 µg kg(-1) , given twice intraperitoneally) or melatonin (10 or 25 mg kg(-1) x 2) the lipopolysaccharide-induced response was elevated 4.6- and 3.5-folds at the most. The cholecystokinin-A receptor antagonist lorglumide reduced the inhibitory effect of cholecystokinin-8, while the melatonin 2-preferring receptor antagonist luzindole had no effect on the melatonin-induced inhibition. Unselective nitric oxide-synthase inhibition abolished the increase in myeloperoxidase activity, whereas inhibition of inducible or neuronal nitric oxide-synthase (of non-nervous origin) halved the inflammatory response. CONCLUSION: Some hormones may contribute to anti-inflammatory action in salivary glands in physiological conditions. They are potential pharmacological tools for treating gland inflammation. The inflammation, as judged from the myeloperoxidase activity, was entirely dependent on nitric oxide-synthase activity, indicating that the hormones directly or indirectly reduced the generation of nitric oxide.

Repetitive non-thermal melting as a timing monitor for femtosecond pump/probe X-ray experiments.

Time-resolved optical pump/X-ray probe experiments are often used to study structural dynamics. To ensure high temporal resolution, it is necessary to monitor the timing between the X-ray pulses and the laser pulses. The transition from a crystalline solid material to a disordered state in a non-thermal melting process can be used as a reliable timing monitor. We have performed a study of the non-thermal melting of InSb in single-shot mode, where we varied the sample temperature in order to determine the conditions required for repetitive melting. We show how experimental conditions affect the feasibility of such a timing tool.

Altered plasticity of the parasympathetic innervation in the recovering rat submandibular gland following extensive atrophy.

Adult rat submandibular glands have a rich autonomic innervation, with parasympathetic and sympathetic nerves working in synergy rather than antagonistically. Ligation of the secretory duct rapidly causes atrophy and the loss of most acini, which are the main target cell for parasympathetic nerves. Following deligation, there is a recovery of gland structure and function, as assessed by autonomimetic stimulation. This study examines whether the parasympathetic nerves reattach to new target cells to form functional neuro-effector junctions. Under recovery anaesthesia, the submandibular duct of adult male rats was ligated via an intra-oral approach to avoid damaging the chorda-lingual nerve. Four weeks later, rats were either killed or anaesthetized and the ligation clip removed. Following a further 8 weeks, both submandibular ducts were cannulated under terminal anaesthesia. Salivary flows were then stimulated electrically (chorda-lingual nerve at 2, 5 and 10 Hz) and subsequently by methacholine (whole-body infusion at two doses). Glands were excised, weighed and divided for further in vitro studies or fixed for histological examination. Ligation of ducts caused 75% loss of gland weight, with the loss of most acinar cells. Of the remaining acini, only 50% were innervated despite unchanged choline acetyltransferase activity, suggesting few parasympathetic nerves had died. Following deligation, submandibular glands recovered half their weight and had normal morphology. Salivary flows from both glands (per unit of gland tissue) were similar when evoked by methacholine but greater from the deligated glands when evoked by nerve stimulation. This suggests that parasympathetic nerves had reattached to new target cells in the recovered glands at a greater ratio than normal, confirming reinnervation of the regenerating gland.

Generation of a large compressive strain wave in graphite by ultrashort-pulse laser irradiation.

We have studied strain wave generation in graphite induced by an intense ultrashort laser pulse. The study was performed in the intensity regime above the ablation threshold of graphite. The aim was to maximize the strain and, thus, also the internal pressure (stress). Laser pulses with a 1 ps temporal duration melt the surface of graphite resulting in a molten material which initially exists at the solid density. As the molten material expands, a compressive strain wave starts propagating into the crystal below the molten layer. The strain pulse was studied with time-resolved X-ray diffraction. At a temporal delay of 100 ps after laser excitation, we observed >10% compressive strain, which corresponds to a pressure of 7.2 GPa. This strain could be reproduced by hydrodynamic simulations, which also provided a temperature map as a function of time and depth.