Improved ex vivo fluorescence imaging of human head and neck cancer using the peptide tracer TPP-IRDye800 targeting membrane-bound Hsp70 on tumor cells.

BACKGROUND: The primary goal of surgery in HNSCC is the complete resection of tumor cells with maximum preservation of normal tissue. The membrane Hsp70-targeting fluorescence labelled peptide TPP-IRDye800 represents a promising tool for real-time intraoperative tumor visualization, enabling the detection of true tumor margins, critical isles of high-grade dysplasia and LN metastases. METHODS: Membrane Hsp70 (mHsp70) expression on HNSCC cell lines and primary HNSCC was determined by flow cytometry and fluorescence microscopy using FITC-conjugated mAb cmHsp70.1 and TPP. TPP-IRDye800 was sprayed on freshly resected tumor material of immunohistochemically confirmed HNSCC and LN metastases for tumor imaging. TBRs were compared using TPP-IRDye800 and Cetuximab-IRDye680, recognizing EGFR. RESULTS: mHsp70 expressing HNSCC cells specifically bind and internalize TPP in vitro. The TBR (2.56 ± 0.39) and AUC [0.98 CI, 0.95-1.00 vs. 0.91 CI, 0.85-0.97] of TPP-IRDye800 on primary HNSCC was significantly higher than Cetuximab-IRDye680 (1.61 ± 0.39) (p = 0.0068) and TPP-IRDye800 provided a superior tumor delineation. Fluorescence imaging showed higher AUC values than a visual inspection by surgeons [0.97 CI, 0.94-1.00 vs. 0.92 CI, 0.88-0.97] (p = 0.048). LN metastases could be visualized using TPP-IRDye800. Real-time tissue delineation was confirmed using the clinically applied KARL-STORZ imaging system. CONCLUSION: TPP-IRDye800 is a promising fluorescence imaging probe for HNSCC.

Cranial autonomic symptoms in migraine are related to central sensitization: a prospective study of 164 migraine patients at a tertiary headache center.

BACKGROUND: Cranial autonomic symptoms (CASs) during migraine attacks are reported to be quite common regardless of ethnicity. In our previous study investigating 373 migraineurs, we found that 42.4% of them had CASs. The patients with CASs more frequently had cutaneous allodynia than did those without CASs, and we speculated that CASs were associated with central sensitization. The present study searched for substantial evidence on the relationship between CASs and central sensitization in migraine patients. METHODS: This was a prospective cross-sectional study. We studied a new independent cohort of 164 migraineurs who presented to the Tominaga Hospital Headache Center from July 2018 until December 2019. The clinical features of CASs according to the criteria in ICHD-3 (beta) were investigated. We also evaluated central sensitization based on the 25 health-related symptoms utilizing the validated central sensitization inventory (CSI), and each symptom was rated from 0 to 4 resulting a total score of 0-100. RESULTS: The mean age was 41.8 (range: 20 to 77) years old. One hundred and thirty-one patients (78.9%) were women. Eighty-six of the 164 (52.4%) patients had at least 1 cranial autonomic symptom. The CSI score of the patients with ≥3 CASs reflected a moderate severity and was significantly higher than in those without CASs (41.9 vs. 30.7, p = 0.0005). The score of the patients with ≥1 conspicuous CAS also reflected a moderate severity and was significantly higher than in those without CASs (40.7 vs. 33.2, p = 0.013). The patients in the CSI ≥40 group had lacrimation, aural fullness, nasal blockage, and rhinorrhea, which are cranial autonomic parasympathetic symptoms, significantly more frequently than those in the CSI < 40 group. CONCLUSIONS: Migraine patients with CASs showed significantly greater central sensitization than those without such symptoms. In particular, cranial parasympathetic symptoms were more frequent in centrally sensitized patients than in nonsensitized patients, suggesting that cranial parasympathetic activation may contribute to the maintenance of central sensitization. TRIAL REGISTRATION: This study was retrospectively registered with UMIN-CTR on 29 Aug 2020 ( UMIN000041603 ).

Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination.

BACKGROUND: Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology. RESULTS: For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci. CONCLUSIONS: Our findings provide a novel efficient approach to promote precise gene editing in human cells.

The relation between emotion regulation and migraine: A cross-cultural study on the moderating effect of culture.

BACKGROUND: Effects of emotion suppression on physical health might be contingent on culture. Existing research on emotion regulation has mainly included western participants. Herewith the question arises, whether this gained expertise is transferable to an Asian culture. OBJECTIVES: This cross-sectional study evaluated to what extent the regulation of emotions is related to migraine and if the relation between emotion regulation and migraine complaints differs between a Western and an Asian population. Therefore, the main characteristics and symptoms of patients with migraine from both Germany and Japan are compared. METHODS: 261 Japanese and 347 German headache patients participated in this online study and completed self-report measures of emotion regulation (suppression and reappraisal) and headache complaints. RESULTS: Cultural groups did not differ regarding their demographic data, intake of medication and number of days with headache. German participants showed significantly higher levels of anxiety and lower levels of emotion suppression compared to Japanese patients. Emotion regulation is not correlated with headache complaints either in the Japanese or in the German patient group. CONCLUSION: Although group differences were found with respect to anxiety and emotion suppression, subsequent regression analysis revealed these differences were unrelated to headache complaints. As our baseline analysis focused on group means, approaches that examine individual reaction patterns to stress and accompanying sensory stimulus processing may prove to be more fruitful and illuminating.

Cranial Autonomic Symptoms of Migraine in Japan: Prospective Study of 373 Migraine Patients at a Tertiary Headache Center.

OBJECTIVE: To clarify the detailed clinical characteristics of cranial autonomic symptoms (CAS) of Japanese patients with migraine and to get insight into the pathophysiological implications. BACKGROUND: Recent studies reported that CAS in migraine is causing diagnostic confusion with cluster headache or sinus headache; however, most reports have concerned Caucasians, and Asian data are scarce. The regional differences in the clinical characteristics of primary headaches between Caucasians and Asians have also been revealed recently. METHOD: This was a cross-sectional study. We investigated 373 patients with migraine in a tertiary headache center with face-to-face interviews. RESULTS: According to our findings, 158/373 (42.4%) patients with migraine had CAS and were characterized by more frequent cutaneous allodynia than those without CAS, suggesting the contribution of central sensitization; however, there were no statistically significant differences in pulsating pain or motion sensitivity as signs of peripheral sensitization. In contrast to the previous study, osmophobia was found to be significantly related to CAS. CONCLUSION: CAS in patients with migraine is common not only in Caucasians but also in Asians. Central sensitization seems to contribute more than peripheral sensitization to CAS manifestation, and osmophobia might be noteworthy among Asian patients with migraine. To avoid a misdiagnosis, we emphasize the need for comments on CAS in the international classification of headache disorders migraine criteria.

Dual-comb spectroscopy using plasmon-enhanced-waveguide dispersion-compensated quantum cascade lasers.

In this Letter, we report on sub-millisecond response time mid-infrared dual-comb spectroscopy using a balanced asymmetric (dispersive) dual-comb setup with a matched pair of plasmon-enhanced-waveguide dispersion-compensated quantum cascade lasers. The system performance is demonstrated by measuring spectra of Bromomethane (CH3Br) and Freon 134a (CH2FCF3) at approximately 7.8 µm. A purely computational phase and timing-correction procedure is used to validate the coherence of the quantum cascade lasers frequency combs and to enable coherent averaging over the time scales investigated. The system achieves a noise-equivalent absorption better than 1×10-3 Hz-1/2, with a resolution of 9.8 GHz (0.326 cm-1) and an optical bandwidth of 1 THz (32 cm-1), with an average optical power of more than 1 mW per spectral element.

3.36 µm single-mode quantum cascade laser with a dissipation below 250 mW.

We present 3.36 µm buried heterostructure distributed-feedback quantum cascade lasers with a power dissipation at threshold below 250 mW and operation temperatures as high as 130 °C. Threshold values below 20 mA at -10 °C in pulsed operation and 30 mA at -20 °C in continuous-wave operation are reported. Optical power above 130 mW and 13 mW are achieved at -20 °C in pulsed and continuous-wave operation, respectively. Continuous-wave operation occurs until 15 °C. We show single-mode emission in pulsed and continuous-wave operation. Single-mode performance is demonstrated in long pulse (5.56 µs) operation. The laser far-field exhibits a single lobe emission with full-width-half-max of 27 ° × 34 °.

Extended tuning of mid-ir quantum cascade lasers using integrated resistive heaters.

We present single mode quantum cascade lasers including a microscopic heater for spectral emission tuning. Through the use of a buried heater element, the active region temperature can be modified without changing the submount one. Emission frequency tuning in continuous-wave as large as 9 cm(-1) at 1270 cm(-1) and 14 cm(-1) at 2040 cm(-1) are observed, corresponding to an increase of the active region temperatures of ∼ 90 K. Due to the proximity of the heaters to the active region, emission can be modulated at several kHz range and the absence of moving parts guarantees the mechanical stability of the system. This method can be successfully applied to all buried heterostructure lasers, becoming an attractive solution for molecular spectroscopy in the IR. Using the presented devices, molecular absorptions of N(2)O have been measured between 1270 cm(-1) and 1280 cm(-1) and are in agreement with data from the HITRAN database.

Impact of interface roughness distributions on the operation of quantum cascade lasers.

We study the impact of interface roughness on the operation of mid-IR and THz quantum cascade lasers. Particular emphasis is given towards the differences between the Gaussian and exponential roughness distribution functions, for which we present results from simulation packages based on nonequilibrium Green's functions and density matrices. The Gaussian distribution suppresses scattering at high momentum transfer which enhances the lifetime of the upper laser level in mid-IR lasers. For THz lasers, a broader range of scattering transitions is of relevance, which is sensitive to the entire profile of the interface fluctuations. Furthermore we discuss the implementation of interface roughness within a two band model.

Distributed-feedback quantum cascade laser emitting at 3.2 µm.

In this work, we present GaInAs/AlAs/AlInAs quantum cascade lasers emitting from 3.2 to 3.4 µm. Single-mode emission is obtained using buried distributed-feedback gratings fabricated using standard deep-UV contact lithography. This technique can easily be transferred to industrial production. Devices with single-mode emission down to 3.19 µm were achieved with peak power of up to 250 mW at -20 °C. A tuning range of 11 cm(-1) was obtained by changing the device temperature between -30 °C and 20 °C.

The effect of ε-aminocaproic acid on blood product requirement, outcome and thromboelastography parameters in severely thrombocytopenic dogs.

BACKGROUND: No treatment other than platelet administration is known to protect against spontaneous hemorrhage in thrombocytopenic dogs. OBJECTIVES: Primary: determine if treatment with ε-aminocaproic acid (EACA) decreases the requirement for blood transfusions and improves outcome in dogs with severe thrombocytopenia. Secondary: find evidence of hyperfibrinolysis and determine the effect EACA administration on rapid (rTEG) and tissue plasminogen activator-spiked (tPA-rTEG) thromboelastography parameters. ANIMALS: Twenty-seven dogs with severe thrombocytopenia were treated with EACA, and data from an additional 33 were obtained from the hospital database as historical control (HC) cohort. METHODS: Single arm clinical trial with HCs. The EACA group dogs received EACA (100 mg/kg IV followed by a constant-rate infusion [CRI] of 400 mg/kg/24 hours). Thromboelastography before and during EACA infusion, hospitalization days, number of transfusions, and mortality were compared. RESULTS: No difference was found in number of transfusions per dog (median, interquartile range; 1, 0-2.5 vs 0.9, 0-2; P = .5) and hospitalization days (4, 4-6 vs 4.5, 3.75-6; P = .83) between HC and EACA groups, respectively, and no difference in survival was identified by log-rank analysis (P = .15). Maximum amplitude on both rTEG and tPA-rTEG increased after EACA administration (rTEG baseline: 23.6, 9.6-38.9; post-EACA: 27.3, 19.8-43.2; P < .001; tPA-rTEG baseline: 23, 10.9-37.2; post-EACA: 24.7, 16.7-44.8; P < .002). CONCLUSIONS AND CLINICAL IMPORTANCE: Although EACA increased clot strength, there was no effect on outcome. Treatment with EACA at this dosage cannot be recommended as a routine treatment but may be considered for dogs with severe ongoing hemorrhage.

Diffuse optical spectroscopy of lactating and non-lactating human mammary physiology.

Breastfeeding provides widely recognized advantages for infant and maternal health. Unfortunately, many women experience trouble with breastfeeding. Nevertheless, few suitable imaging modalities are available to study human lactation and determine the possible causes of breastfeeding problems. In this study, we apply broadband, quantitative diffuse optical spectroscopy (DOS) for this purpose. We present a study of fourteen lactating and eight similarly aged, premenopausal, non-lactating women to investigate the feasibility of DOS to study the optical and physiological differences between 1) lactating and non-lactating breasts, 2) the areolar and non-areolar region within the breast, and 3) lactating breasts before and after milk extraction. Our study shows that i) the median total hemoglobin concentration [tHb] of the lactating breast is 51% higher than for the non-lactating breast. ii) the median [tHb] of the lactating breast is 37% higher in the areolar region compared to the non-areolar region. iii) lactating breasts exhibit a positive median difference of 8% in [tHb] after milk extraction. Our findings are consistent with the expected physiological changes that occur during the lactation period. Importantly, we show that DOS provides unique insight into breast tissue composition and physiology, serving as a foundation for future application of the technique in lactation research.

Biosynthesis of the allelopathic alkaloid gramine in barley by a cryptic oxidative rearrangement.

The defensive alkaloid gramine not only protects barley and other grasses from insects but also negatively affects their palatability to ruminants. The key gene for gramine formation has remained elusive, hampering breeding initiatives. In this work, we report that a gene encoding cytochrome P450 monooxygenase CYP76M57, which we name AMI synthase (AMIS), enables the production of gramine in Nicotiana benthamiana, Arabidopsis thaliana, and Saccharomyces cerevisiae. We reconstituted gramine production in the gramine-free barley (Hordeum vulgare) variety Golden Promise and eliminated it from cultivar Tafeno by Cas-mediated gene editing. In vitro experiments unraveled that an unexpected cryptic oxidative rearrangement underlies this noncanonical conversion of an amino acid to a chain-shortened biogenic amine. The discovery of the genetic basis of gramine formation now permits tailor-made optimization of gramine-linked traits in barley by plant breeding.

Hemiplegic migraine type 2 with new mutation of the ATP1A2 gene in Japanese cases.

We analyzed the clinical symptoms of hemiplegic migraine (HM) and their relevance in four Japanese patients considered to have ATP1A2 mutations as a cause. Sequencing of ATP1A2 was performed using the Sanger method in 43 blood samples from clinically suspected patients with familial HM. Subsequently, algorithm analysis, allele frequency determination, and three-dimensional structure analysis of the recognized variants were performed, and the recognized variants were evaluated. We found four heterozygous missense mutations in ATP1A2 (Case 1: p.R51C; Case 2: p.R65L; Case 3: p.A269P; Case 4: p.D999H), three of which had not been reported to date. These four mutations may also affect the structure of the protein products, as assessed using a three-dimensional structural analysis. In all four cases, the clinical symptoms included visual, sensory, motor, and verbal symptoms and the frequency and duration of headache attacks varied. Additionally, oral administration of a combination of lomerizine hydrochloride and topiramate had a partial effect in three cases. We report four missense mutations in ATP1A2. This report will be useful for the future analysis of mutations and clinical types in Asians, as well as Westerners, with migraine.

Elevated myocardial enzymes and natriuretic peptides in anorexia nervosa: prototypic condition for the pathophysiology of cachexia?

We report on a patient suffering from chronic anorexia nervosa who in the course of treatment showed elevated high-sensitive troponin T, creatine kinase and most markedly N-terminal pro-brain natriuretic peptide (NT-proBNP). Elevated enzymes improved significantly throughout the course of treatment without cardiac specific medication but exceeded the normal range for weeks. Abnormally high myocardial enzymes and NT-proBNP in cachectic anorectic patients might resemble conditions of cardiac cachexia. A review of the available literature is provided. Further research is required to explain the pathophysiological meaning of the abnormal laboratory findings.

Ultrasound-guided paravertebral perineural glucocorticoid injection for signs of refractory cervical pain associated with foraminal intervertebral disk protrusion in four dogs.

CASE DESCRIPTION: 4 dogs, 7.5 to 10 years of age, were presented for evaluation of signs of chronic cervical pain and forelimb lameness secondary to cervical foraminal intervertebral disk protrusion (IVDP). All dogs were refractory to ≥ 2 weeks of conservative management including strict rest and pain management with anti-inflammatory drugs, methocarbamol, and gabapentin. CLINICAL FINDINGS: The MRI findings included left foraminal IVDP at C2-3 causing mild C3 nerve root compression (dog 1), multifocal degenerative disk disease with mild focal left-sided disk protrusion at C6-7 without associated spinal cord or nerve root compression (dog 2), left foraminal C6-7 IVDP with suspected focal spinal cord atrophy or mild compression (dog 3), and right foraminal C6-7 IVDP and multifocal cervical intervertebral disk degeneration with annulus fibrosus protrusion (dog 4). TREATMENT AND OUTCOME: Ultrasound-guided paravertebral perineural injections with methylprednisolone acetate (1 mg/kg [0.45 mg/lb]) at the C3 nerve root in dog 1 and at the C7 nerve root in the other 3 dogs were performed. Injections were repeated at intervals of 4 weeks to 3 months on the basis of clinical response. None of the dogs had any complications from the procedures. For dogs 1 and 4, there was complete resolution of lameness and signs of cervical pain following perineural injections, and for dog 3, there was complete resolution of lameness and only minimal residual cervical pain. Dog 2 did not have long-lasting improvement. CLINICAL RELEVANCE: Findings indicated that ultrasound-guided paravertebral perineural injection can be an effective treatment of cervical foraminal IVDP for some dogs. Additional studies to determine appropriate case selection and better assess the overall success rate and risks associated with this technique are warranted.

Evaluation of the changes in human milk lipid composition and conformational state with Raman spectroscopy during a breastfeed.

Human milk fat forms the main energy source for breastfed infants, and is highly variable in terms of concentration and composition. Understanding the changes in human milk lipid composition and conformational state during a breastfeed can provide insight into lipid synthesis and secretion in the mammary gland. Therefore, the aim of this study was to evaluate human milk fatty acid length, degree of unsaturation (lipid composition) and lipid phase (lipid conformational state) at different stages during a single breastfeed (fore-, bulk- and hindmilk). A total of 48 samples from 16 lactating subjects were investigated with confocal Raman spectroscopy. We did not observe any significant changes in lipid composition between fore-, bulk and hindmilk. A new finding from this study is that lipid conformational state at room temperature changed significantly during a breastfeed, from almost crystalline to almost liquid. This observation suggests that lipid synthesis in the mammary gland changes during a single breastfeed.

End-of-life practices in the Netherlands under the Euthanasia Act.

BACKGROUND: In 2002, an act regulating the ending of life by a physician at the request of a patient with unbearable suffering came into effect in the Netherlands. In 2005, we performed a follow-up study of euthanasia, physician-assisted suicide, and other end-of-life practices. METHODS: We mailed questionnaires to physicians attending 6860 deaths that were identified from death certificates. The response rate was 77.8%. RESULTS: In 2005, of all deaths in the Netherlands, 1.7% were the result of euthanasia and 0.1% were the result of physician-assisted suicide. These percentages were significantly lower than those in 2001, when 2.6% of all deaths resulted from euthanasia and 0.2% from assisted suicide. Of all deaths, 0.4% were the result of the ending of life without an explicit request by the patient. Continuous deep sedation was used in conjunction with possible hastening of death in 7.1% of all deaths in 2005, significantly increased from 5.6% in 2001. In 73.9% of all cases of euthanasia or assisted suicide in 2005, life was ended with the use of neuromuscular relaxants or barbiturates; opioids were used in 16.2% of cases. In 2005, 80.2% of all cases of euthanasia or assisted suicide were reported. Physicians were most likely to report their end-of-life practices if they considered them to be an act of euthanasia or assisted suicide, which was rarely true when opioids were used. CONCLUSIONS: The Dutch Euthanasia Act was followed by a modest decrease in the rates of euthanasia and physician-assisted suicide. The decrease may have resulted from the increased application of other end-of-life care interventions, such as palliative sedation.

Trauma-induced primary hypoparathyroidism following severe bite wound injury to the neck in a dog.

OBJECTIVE: To describe the clinical presentation and outcome of a dog with primary hypoparathyroidism secondary to cervical bite wounds. CASE SUMMARY: A 3-year-old male intact Chihuahua presented after being attacked by a large breed dog. The dog sustained severe cervical lacerations, exposing the trachea and jugular veins. A portion of the right thyroid gland was missing. The dog was stabilized before wound debridement and closure. Ionized calcium concentrations were within reference range at the time of presentation. Forty-eight hours after the initial trauma, the dog was presented in lateral recumbency with signs of hypovolemic shock, muscle tremors, and hyperthermia. Bloodwork showed severe ionized hypocalcemia with low normal parathyroid hormone concentration consistent with acute primary hypoparathyroidism. The dog was managed initially with IV calcium gluconate and calcitriol, then long-term oral calcium carbonate and vitamin D3. After 6 months, the dog was successfully weaned off calcium supplementation. NEW OR UNIQUE INFORMATION PROVIDED: This is the first described case of traumatic primary hypoparathyroidism after a bite injury to the neck in a dog.

Perceptions of heatwave risks to health: interview-based study of older people in London and Norwich, UK.

BACKGROUND: Most projections of climate change suggest an increased frequency of heatwaves in England over coming decades; older people are at particular risk. This could result in substantial mortality and morbidity. OBJECTIVE: To determine elderly people's knowledge and perceptions of heat-related risks to health, and of protective behaviours. METHODS: Semi-structured interviews: 73 men and women, 72-94 years, living in their own homes in London and Norwich, UK. RESULTS: Few respondents considered themselves either old or at risk from the effects of heat, even though many had some form of relevant chronic illness; they did recognize that some medical conditions might increase risks in others. Most reported that they had taken appropriate steps to reduce the effects of heat. Some respondents considered it appropriate for the government to take responsibility for protecting vulnerable people, but many thought state intervention was unnecessary, intrusive and unlikely to be effective. Respondents were more positive about the value of appropriately disseminated advice and solutions by communities themselves. CONCLUSION: The Heatwave Plan should consider giving greater emphasis to a population-based information strategy, using innovative information dissemination methods to increase awareness of vulnerability to heat among the elderly and to ensure clarity about behaviour modification measures.