Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury

Neuronal calcium (Ca²⁺)-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca²⁺-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca²⁺-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca²⁺-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.Calcium (Ca²⁺) plays a crucial role in many and diverse cellular processes, including neurotransmission (1). Glutamate and neuropeptides are neurotransmitters released from the central terminals of dorsal root ganglion (DRG) neurons in the spinal dorsal horn, where signals for different sensory modalities, including pain, are conveyed to higher centers (2–12). Neurotransmitter release is tightly regulated by Ca²⁺-dependent SNARE proteins whose activity is regulated by Ca²⁺-binding proteins (CaBPs) (1, 7, 13).Parvalbumin (PV), calbindin D-28K (CB), calretinin (CR), and secretagogin (Scgn) are extensively studied EF-hand CaBPs, and they have also emerged as valuable anatomical markers for morphologically and functionally distinct neuronal subpopulations (14–17). The expression of CaBPs in DRG neurons has been thoroughly studied (18). Moreover, neuronal Ca²⁺ sensor 1 and downstream regulatory element-antagonist modulator (DREAM) are also EF-hand Ca²⁺-binding proteins in DRGs and the spinal cord (19, 20). Despite these advances, a CaBP has so far not been characterized in the majority of small- and medium-sized DRG neurons, many of which represent nociceptors.The subfamily of neuronal Ca²⁺-binding proteins (NECABs) consists of three members (NECAB1–NECAB3), probably as a result of gene duplication (21). NECABs are also EF-hand proteins, with one pair of EF-hand motifs in the N terminus and a putative antibiotic biosynthesis monooxygenase domain in the C terminus, which are linked by a NECAB homogeneous region (22). NECAB1/2 are restricted to the nervous system, whereas NECAB3 is also expressed in the heart and skeletal muscle (21).NECAB1 was first identified as the target protein of synaptotagmin I C2A-domain by affinity chromatography, with its expression restricted to layer 4 cortical pyramidal neurons, inhibitory interneurons, and hippocampal CA2 pyramidal cells in mouse brain (21, 23). The gene of the second member was cloned from mouse and initially named Necab. It encodes a 389-aa (NECAB2) (24). NECAB2 was identified as a downstream target of Pax6 in mouse retina, which is involved in retinal development (24, 25), as well as being a binding partner for the adenosine A_2A receptor (22). Furthermore, an interaction between NECAB2 and metabotropic glutamate receptor 5 (mGluR5) was demonstrated in rat hippocampal pyramidal cells, possibly regulating mGluR5’s coupling to its signaling machinery (26). Finally, NECAB3, also known as XB51, was isolated as an interacting target for the neuron-specific X11-like protein and is possibly involved in the pathogenesis of Alzheimer’s disease (27, 28).Very recently, NECAB1/2 were shown to have complementary expression patterns in mouse hippocampus at the mRNA and protein levels, whereas NECAB3 is broadly distributed in the hippocampus (29). NECAB1-expressing cells were seen throughout the cell-sparse layers of Ammon’s horn and the hilus of the dentate gyrus. In contrast, NECAB2 is enriched in pyramidal cells of the CA2 region. A minority of NECAB1⁺ neurons were GABAergic yet did not coexpress PV, CB, or CR (29).Here, we investigated the expression of NECAB1/2 in mouse DRGs and spinal cord using quantitative PCR (qPCR), immunohistochemistry (also combined with CLARITY) (30), and Western blotting. We compared the distribution of NECABs with that of the four CaBPs restricted to neurons, PV, CB, CR, or Scgn. NECAB⁺ neurons in the spinal dorsal horn were phenotyped using transgenic mice harboring genetic markers for excitatory [vesicular glutamate transporter 2 (VGLUT2)] (31) or inhibitory [glutamate decarboxylase 67 (GAD67)] (32) cell identities. Finally, the effect of peripheral nerve injury was analyzed.     

医疗器械可沥滤物安全性研究（三）：允许限量建立

随着新材料、新设计等创新性医疗器械产品的不断涌现,可沥滤物的安全性研究也面临新的挑战,其中可沥滤物允许限量的建立是医疗器械安全性评价研究的重要方面。结合现有产品的审评,简述高分子医疗器械可沥滤物安全性研究中允许限量的建立环节中的常见问题,并对其原因进行解析,供相关机构及研发人员参考使用。     

Analysis of the myoelectric characteristics of genioglossus in REM sleep and its improvement by CPAP treatment in OSA patients

Sleep and Breathing - To reveal the characteristics of genioglossus (GG) activation in moderate and severe obstructive sleep apnea (OSA) patients during rapid eye movement (REM) sleep compared with...     

NPM1, FLT3-ITD,CEBPA, and c-kit mutations in 312 Chinese patients with de novo acute myeloid leukemia

Objectives

To explore NPM1, FLT3-ITD, CEBPA, and c-kit mutations in patients with acute myeloid leukemia (AML) from Chinese population.

Methods

In this study, we retrospectively analyzed the prevalence and clinical pro?le of NPM1, FLT3-ITD, CEBPA, and c-kit mutations in 312 patients with de novo AML.

Results

The frequencies of NPM1, FLT3-ITD, c-kit, and CEBPA mutations were 15.4, 14.0, 7.64, and 25.6%, respectively. The occurrence rate of NPM1 mutations increased with age in patients younger than 60 years. NPM1, c-kit, and CEBPA mutations were all associated with French-American-British subtypes. Patients with NPM1 mutations and FLT3-ITD presented with higher peripheral white blood cell counts and marrow blast percentages.

Conclusion

Both this and previous studies may suggest low frequencies of NPM1 and FLT3-ITD mutations in AML patients from the Chinese population, and they may have a synergistic function in stimulating proliferation of leukemia cells.     

Noninvasive central pulse pressure is an independent determinant of renal function

The purpose of this study was to investigate the prognostic properties of different BP measurements for renal function decrement and early chronic kidney disease (CKD) in community‐dwelling populations with normal renal function at baseline. A total of 1426 participants were included and followed for a median of 4.8 years (interquartile range, 4.5‐5.2), and central hemodynamic profile and estimated glomerular filtration rate (eGFR) were evaluated. One main outcome was the rapid eGFR decline defined as a decline in eGFR of greater than 3 mL/min per 1.73 m² per year; the other was the new incidence of CKD. At the end of follow‐up, mean eGFR decreased from 93.39 ± 13.46 mL/min per 1.73 m² to 85.72 ± 14.81 mL/min per 1.73 m², and the incidence of rapid eGFR decline and CKD were 20.7% and 5.6%, respectively. In multivariate linear regression analysis, central pulse pressure (PP), age, fasting blood glucose, and concentration of homocysteine were independent determinants of the change in renal function. Not only in the prediction of rapid eGFR decline but also in the incident of CKD, baseline central PP was the only BP component that consistently independently associated with both outcomes after adjustment for various confounders. When compared with subjects in the lowest quartile of central PP, those in the highest quartile demonstrated a significantly increased risk of CKD (hazard ratio [HR], 1.57; 95% confidence interval [CI], 1.08‐2.96; P = .006). The study showed that central PP emerged as an independent predictor of the decline in renal function.     

Peak blood pressure‐guided monitoring may serve as an effective approach for blood pressure control in the out‐of‐office setting

We aimed to explore whether diurnal blood pressure (BP) peak characteristics have a significant influence on the association between left ventricular damage with the two BP components (morning BP vs. afternoon peak BP) in untreated hypertensives. This cross‐sectional study included 1084 hypertensives who underwent echocardiography and 24‐h ambulatory BP monitoring. Participants were stratified according to the relationship between morning systolic BP (MSBP; average SBP within 2 h of waking up) and afternoon peak systolic BP (ASBP; average SBP between 16:00 and 18:00). Afternoon and morning hypertension was defined as ≥ 135/85 mm Hg. The morning and afternoon peak BPs occurred at around 7:00 and 17:00, respectively. In general hypertensives, morning BP and afternoon peak BP are significantly different in absolute values (for binary SBP, McNemar''s χ² = 6.42; p = .014). ASBP was more pronounced than MSBP in 602 patients (55.5%), in whom 24‐h SBP showed higher consistency with ASBP than with MSBP (Kappa value: 0.767 vs 0.646, both p < .01). In subjects with ASBP ≥ MSBP, ASBP was associated with left ventricular hypertrophy independent of MSBP (logistic regression analysis odds ratio: 1.046, p < .01), and left ventricular mass index was more strongly correlated with ASBP than with MSBP (multiple regression coefficient β: 0.453, p < .01), in which the relationships held true independently of 24‐h SBP. The opposite results were obtained in subjects with MSBP > ASBP. Peak BP‐guided monitoring may serve as an effective approach to out‐of‐office hypertension monitoring and control, providing the best consistency with 24‐h average SBP and highest discrimination performance for target organ damage, independently of 24‐h SBP.     

Relationship between high‐normal albuminuria and arterial stiffness in Chinese population

High‐normal albuminuria is related to the morbidity and mortality of cardiovascular disease. Arterial stiffness has been regarded as a predictor of cardiovascular disease. However, the relationship between high‐normal albuminuria and arterial stiffness is uncertain in Chinese population. A total of 1343 Chinese participants (aged 58.9 ± 12.1 years, 63.53% male) were included in this study. High‐normal albuminuria was defined as urinary albumin‐to‐creatinine ratio (UACR) above the median within normal albuminuria. Based on the level of UACR, all participants were divided into low‐normal albuminuria group (UACR < 6.36 mg/g, n = 580), high‐normal albuminuria group (6.36 mg/g ≤ UACR < 30 mg/g, n = 581), microalbuminuria (30 mg/g ≤ UACR < 300 mg/g, n = 162), and macroalbuminuria (UACR ≥ 300 mg/g, n = 20). Arterial stiffness was assessed by measuring carotid‐femoral pulse wave velocity (cfPWV). With the increment of UACR, the level of cfPWV was increased gradually (P < .001). Stepwise multiple regression analysis showed that systolic blood pressure, age, serum creatinine, heart rate, logarithmic (LG)‐transformed UACR, and fasting plasma glucose were independently associated with cfPWV in all subjects (P < .001). LG‐UACR was found to be related to cfPWV in high‐normal albuminuria and macroalbuminuria subjects. After further stratification in the high‐normal albuminuria subjects, their relation remained in male, elderly over 65 years old, or normotensives. In summary, UACR is associated with arterial stiffness in subjects with proteinuria excretion in high normal level. High‐normal albuminuria might be an early indicator of arterial stiffness, especially in male, elderly, or normotensives in Chinese population. Furthermore, age and blood pressure are still observed to be the most important risk factor of arterial stiffness.