Inhibition of SIRT7 overcomes sorafenib acquired resistance by suppressing ERK1/2 phosphorylation via the DDX3X-mediated NLRP3 inflammasome in hepatocellular carcinoma.

AIMS: Sirtuin 7 (SIRT7) plays an important role in tumor development, and has been characterized as a potent regulator of cellular stress. However, the effect of SIRT7 on sorafenib acquired resistance remains unclear and a possible anti-tumor mechanism beyond this process in HCC has not been clarified. We examined the therapeutic potential of SIRT7 and determined whether it functions synergistically with sorafenib to overcome chemoresistance. METHODS: Cancer Genome Atlas-liver HCC data and unbiased gene set enrichment analyses were used to identify SIRT7 as a potential effector molecule in sorafenib acquired resistance. Two types of SIRT7 chemical inhibitors were developed to evaluate its therapeutic properties when synergized with sorafenib. Mass spectrometry was performed to discover a direct target of SIRT7, DDX3X, and DDX3X deacetylation levels and protein stability were explored. Moreover, an in vivo xenograft model was used to confirm anti-tumor effect of SIRT7 and DDX3X chemical inhibitors combined with sorafenib. RESULTS: SIRT7 inhibition mediated DDX3X depletion can re-sensitize acquired sorafenib resistance by disrupting NLRP3 inflammasome assembly, finally suppressing hyperactive ERK1/2 signaling in response to NLRP3 inflammasome-mediated IL-1ß inhibition. CONCLUSIONS: SIRT7 is responsible for sorafenib acquired resistance, and its inhibition would be beneficial when combined with sorafenib by suppressing hyperactive pro-cell survival ERK1/2 signaling.

Adnp-mutant mice with cognitive inflexibility, CaMKIIα hyperactivity, and synaptic plasticity deficits.

ADNP syndrome, involving the ADNP transcription factor of the SWI/SNF chromatin-remodeling complex, is characterized by developmental delay, intellectual disability, and autism spectrum disorders (ASD). Although Adnp-haploinsufficient (Adnp-HT) mice display various phenotypic deficits, whether these mice display abnormal synaptic functions remain poorly understood. Here, we report synaptic plasticity deficits associated with cognitive inflexibility and CaMKIIα hyperactivity in Adnp-HT mice. These mice show impaired and inflexible contextual learning and memory, additional to social deficits, long after the juvenile-stage decrease of ADNP protein levels to ~10% of the newborn level. The adult Adnp-HT hippocampus shows hyperphosphorylated CaMKIIα and its substrates, including SynGAP1, and excessive long-term potentiation that is normalized by CaMKIIα inhibition. Therefore, Adnp haploinsufficiency in mice leads to cognitive inflexibility involving CaMKIIα hyperphosphorylation and excessive LTP in adults long after its marked expressional decrease in juveniles.

Early correction of synaptic long-term depression improves abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice.

Extensive evidence links Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.

Protein interactome and cell-type expression analyses reveal that cytoplasmic FMR1-interacting protein 1 (CYFIP1), but not CYFIP2, associates with astrocytic focal adhesion.

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.

Sequence preference and structural heterogeneity of BZ junctions.

BZ junctions, which connect B-DNA to Z-DNA, are necessary for local transformation of B-DNA to Z-DNA in the genome. However, the limited information on the junction-forming sequences and junction structures has led to a lack of understanding of the structural diversity and sequence preferences of BZ junctions. We determined three crystal structures of BZ junctions with diverse sequences followed by spectroscopic validation of DNA conformation. The structural features of the BZ junctions were well conserved regardless of sequences via the continuous base stacking through B-to-Z DNA with A-T base extrusion. However, the sequence-dependent structural heterogeneity of the junctions was also observed in base step parameters that are correlated with steric constraints imposed during Z-DNA formation. Further, circular dichroism and fluorescence-based analysis of BZ junctions revealed that a base extrusion was only found at the A-T base pair present next to a stable dinucleotide Z-DNA unit. Our findings suggest that Z-DNA formation in the genome is influenced by the sequence preference for BZ junctions.

Lrfn2-Mutant Mice Display Suppressed Synaptic Plasticity and Inhibitory Synapse Development and Abnormal Social Communication and Startle Response.

SALM1 (SALM (synaptic adhesion-like molecule), also known as LRFN2 (leucine rich repeat and fibronectin type III domain containing), is a postsynaptic density (PSD)-95-interacting synaptic adhesion molecule implicated in the regulation of NMDA receptor (NMDAR) clustering largely based on in vitro data, although its in vivo functions remain unclear. Here, we found that mice lacking SALM1/LRFN2 (Lrfn2-/- mice) show a normal density of excitatory synapses but altered excitatory synaptic function, including enhanced NMDAR-dependent synaptic transmission but suppressed NMDAR-dependent synaptic plasticity in the hippocampal CA1 region. Unexpectedly, SALM1 expression was detected in both glutamatergic and GABAergic neurons and Lrfn2-/- CA1 pyramidal neurons showed decreases in the density of inhibitory synapses and the frequency of spontaneous inhibitory synaptic transmission. Behaviorally, ultrasonic vocalization was suppressed in Lrfn2-/- pups separated from their mothers and acoustic startle was enhanced, but locomotion, anxiety-like behavior, social interaction, repetitive behaviors, and learning and memory were largely normal in adult male Lrfn2-/- mice. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, and social communication and startle behaviors in mice.SIGNIFICANCE STATEMENT Synaptic adhesion molecules regulate synapse development and function, which govern neural circuit and brain functions. The SALM/LRFN (synaptic adhesion-like molecule/leucine rich repeat and fibronectin type III domain containing) family of synaptic adhesion proteins consists of five known members for which the in vivo functions are largely unknown. Here, we characterized mice lacking SALM1/LRFN2 (SALM1 KO) known to associate with NMDA receptors (NMDARs) and found that these mice showed altered NMDAR-dependent synaptic transmission and plasticity, as expected, but unexpectedly also exhibited suppressed inhibitory synapse development and synaptic transmission. Behaviorally, SALM1 KO pups showed suppressed ultrasonic vocalization upon separation from their mothers and SALM1 KO adults showed enhanced responses to loud acoustic stimuli. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, social communication, and acoustic startle behavior.

A Case of Juvenile Psammomatoid Ossifying Fibroma on Skull Base.

Juvenile psammomatoid ossifying fibroma (JPOF) is a rare tumor that occurs in maxillary sinus or orbit. Complete removal is required due to the aggressive and locally destructive nature. It is hard to distinguish from psammomatoid meningioma in cranial lesion and to remove completely. The authors are presenting a case of 26-year-old male with JPOF on skull base and report this case with review of literature.

Structural and functional study of ChuY from Escherichia coli strain CFT073.

The uropathogenic Escherichia coli strain CFT073 contains multiple iron and heme transport systems, which facilitate infection of the host urinary tract. To elucidate the molecular and cellular function of ChuY, a hypothetical gene in the heme degradation/utilization pathway, we solved the crystal structure of ChuY at 2.4 Å resolution. ChuY has high structural homology with human biliverdin and flavin reductase. We confirmed that ChuY has flavin mononucleotide (FMN) reductase activity, using NAD(P)H as a cofactor, and shows porphyrin ring binding affinity. A chuY deletion-insertion strain showed reduced survival potential compared to wild-type and complemented strains in mammalian cells. Current results suggest ChuY acts as a reductase in heme homeostasis to maintain the virulence potential of E. coli CFT073.

Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ).

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the ß-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the ß-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.

Treatment Strategy for Revision Rhinoplasty in Asians.

Revision rhinoplasty in Asian patients is associated with problems related to the use of grafts or implant materials. Moreover, the septal cartilage of Asian individuals is generally weak and small, which makes it particularly vulnerable to injury or secondary deformity during primary surgery. Hence, there is an increased demand for major reconstruction of the septal cartilage framework during revision surgery in Asian patients. In revision rhinoplasty of the nose in Asian patients, appropriate management of the graft or implant is vital. The common problems resulting in the need for revision surgery include displacement, malposition, extrusion, recurrent inflammation, and infection of dorsally implanted alloplastic material. A short-nose deformity following silicone rhinoplasty is also a common problem that is difficult to manage. Furthermore, residual or recurrent deviation of the deviated nose, undercorrection of the convex nasal dorsum, and tip graft-related complications are frequently encountered problems that require revision. In revision rhinoplasty for Asian patients, autologous tissues, such as conchal cartilage and costal cartilage, play a pivotal role for use as a new dorsal implant or building block for major septal reconstruction. Therefore, it is imperative for surgeons to familiarize themselves with the appropriate use of autologous tissues, particularly costal cartilage.

Effects of Elastic Band Resistance Training on Glucose Control, Body Composition, and Physical Function in Women With Short- vs. Long-Duration Type-2 Diabetes.

This study examined whether the existing duration of type-2 diabetes influenced patient responses to progressive resistance training. Twenty-six women with type-2 diabetes were stratified into short- (3 ± 2 years; n = 12) or long-standing (10 ± 3 years; n = 14) disease groups. Patients participated in a high daily or high weekly frequency elastic band resistance training program that consisted of 2 daily sessions, 5 d·wk for 12 weeks. Glucose control, body composition, and physical function were evaluated pre- and posttraining. No significant diabetes duration × training interactions were detected for blood markers of glucose control (p > 0.05); however, there were significant main effects of training driven by comparable improvements in both cohorts (hemoglobin A1c, -13 to 18%; fasting glucose, -23 to 31%; postprandial glucose, -36 to 40%; insulin, -34 to 40%; C-peptide, -38 to 51%; p ≤ 0.05). Anthropometrics and body composition were also favorably modified in both the groups after training (weight, -5 to 9%; body mass index, -6 to 9%; waist-to-hip ratio, -3 to 5%; percent fat, -14 to 20%; p ≤ 0.05). Likewise, indices of physical function improved in both the groups after training (bicep curl repetitions, +15-33%; sit-and-stand repetitions, +45-47%; p ≤ 0.05). A few exceptions were noted in which patients with long-standing disease demonstrated greater pre-to-post gains (p ≤ 0.05) in grip strength (+11-13%) and peak exercise time (+19%) and load (+21%) during graded exercise, whereas those with shorter disease duration did not. Overall, these data suggest that patients with a long history of diabetes respond positively to resistance training and in a manner comparable to their recently diagnosed counterparts. Therefore, current inactivity in patients with long-standing disease should not deter from beginning an exercise program.

De novo mutations in the motor domain of KIF1A cause cognitive impairment, spastic paraparesis, axonal neuropathy, and cerebellar atrophy.

KIF1A is a neuron-specific motor protein that plays important roles in cargo transport along neurites. Recessive mutations in KIF1A were previously described in families with spastic paraparesis or sensory and autonomic neuropathy type-2. Here, we report 11 heterozygous de novo missense mutations (p.S58L, p.T99M, p.G102D, p.V144F, p.R167C, p.A202P, p.S215R, p.R216P, p.L249Q, p.E253K, and p.R316W) in KIF1A in 14 individuals, including two monozygotic twins. Two mutations (p.T99M and p.E253K) were recurrent, each being found in unrelated cases. All these de novo mutations are located in the motor domain (MD) of KIF1A. Structural modeling revealed that they alter conserved residues that are critical for the structure and function of the MD. Transfection studies suggested that at least five of these mutations affect the transport of the MD along axons. Individuals with de novo mutations in KIF1A display a phenotype characterized by cognitive impairment and variable presence of cerebellar atrophy, spastic paraparesis, optic nerve atrophy, peripheral neuropathy, and epilepsy. Our findings thus indicate that de novo missense mutations in the MD of KIF1A cause a phenotype that overlaps with, while being more severe, than that associated with recessive mutations in the same gene.

Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa.

The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co(2+) rather than Zn(2+): the kcat (s(-1)) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co(2+), and Zn(2+)conditions, respectively. Consistently, addition of low concentrations of Co(2+) to PaAP previously saturated with Zn(2+) greatly enhanced the enzymatic activity, suggesting that Co(2+)may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co(2+) or Zn(2+) commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co(2+)- and Zn(2+)-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co(2+) for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.

Distinct Rayleigh scattering from hot spot mutant p53 proteins reveals cancer cells.

The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor.

Hearing gain with a BAHA test-band in patients with single-sided deafness.

PURPOSE: It is assumed that preoperative use of a bone-anchored hearing aid (BAHA) test-band will give a patient lower gain compared to real post-operative gain because of the reduction of energy through the scalp when using a test-band. Hearing gains using a BAHA test-band were analyzed in patients with unilateral hearing loss. MATERIALS AND METHODS: Nineteen patients with unilateral sensorineural hearing loss were enrolled. A test-band, which was connected to BAHA Intenso with full-on gain, was put on the mastoid. Conventional air-conduction (AC) pure-tone averages (PTAs) and sound-field PTAs and speech reception thresholds (SRTs) were obtained in conditions A (the better ear naked), B (the better ear plugged), and C (the better ear plugged with a test-band on the poorer mastoid). RESULTS: Air-conduction PTAs of the poorer and better ears were 91 ± 19 and 18 ± 8 dB HL. Sound-field PTAs in condition B were higher than those in condition A (54 vs. 26 dB HL), which means that earplugs can block the sound grossly up to 54 dB HL through the better ears. The aided PTAs (24 ± 6 dB HL) in condition C were similar to those of the better ears in condition A (26±9 dB HL), though condition C showed higher thresholds at 500 Hz and lower thresholds at 1 and 2kHz when compared to condition A. The hearing thresholds using a test-band were similar to the published results of BAHA users with the volume to most comfortable level (MCL). CONCLUSION: Our findings showed that a BAHA test-band on the poorer ear could transmit sound to the cochlea as much as the better ears can hear. The increased functional gain at 1 and 2kHz reflects the technical characteristics of BAHA processor. The reduction of energy through the scalp when using a test-band seems to be offset by the difference of output by setting the volume to full-on gain and using a high-powered speech processor. Preoperative hearing gains using a test-band with full-on gain seems to be similar to the post-operative gains of BAHA users with the volume to MCL.

Crystallization of Z-DNA in Complex with Chemical and Z-DNA Binding Z-Alpha Protein.

The molecular basis of Z-DNA recognition and stabilization is mostly discovered via X-ray crystallography. The sequences composed with alteration of purine and pyrimidine are known to adopt Z-DNA conformation. Due to the energy penalty for forming Z-DNA, the small molecular stabilizer or Z-DNA-specific binding protein is required for DNA to adopt Z conformation prior to crystallizing Z-DNA. Here we described the methods ranging from preparation of DNA and Z-alpha protein to crystallization of Z-DNA in detail.

BZ Junctions and Its Application as Probe (2AP) to Detect Z-DNA Formation and Its Effector.

The left-handed Z-DNA is surrounded by right-handed canonical B-DNA, and thus the junction between B- and Z-DNA has been occurred during temporal Z-DNA formation in the genome. The base extrusion structure of the BZ junction may help detect Z-DNA formation in DNAs. Here we describe the BZ junction structural detection by using 2-aminopurine (2AP) fluorescent probe. BZ junction formation can be measured in solution by this method.

Suppression of neuroinflammation and α-synuclein oligomerization by rotarod walking exercise in subacute MPTP model of Parkinson's disease.

Parkinson's disease (PD) belongs to an α-synucleinopathy and manifests motor dysfunction attributed to nigrostriatal dopaminergic degeneration. In clinical practice, the beneficial role of physical therapy such as motor skill learning training has been recognized in PD-linked motor defects. Nevertheless, the disease-modifying effects of motor skill learning training on PD-related pathology remain unclear. Here, we investigated the disease-modifying effects of rotarod walking exercise (RWE), a modality of motor skill learning training, in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. In motor function and dopaminergic degeneration, RWE improved MPTP-induced deficits. In addition, RWE enhanced the expression of neurotrophic factors BDNF/GDNF, PGC1-α, Nurr1, and p-AMPK, thereby recovering dopaminergic neuronal cell death. Moreover, RWE inhibited microglial activation and the expression of pro-inflammatory markers, such as p-IκBα, iNOS, IL-1ß, TNF-α, and cathepsin D, while elevating anti-inflammatory IL-10 and TGF-ß. RWE also decreased oxidative stress markers in the substantia nigra, such as 4-HNE and 8-OHdG-positive cells, while increasing Nrf2-controlled antioxidant enzymes. Regarding the effect of RWE on α-synuclein, it reduced the monomer/oligomer forms of α-synuclein and phosphorylation at serine 129. Further mechanistic studies revealed that RWE suppressed the expression of matrix metalloproteinase-3 and p-GSK3ß (Y216), which play key roles in α-synuclein aggregation. These data collectively suggest that inhibition of neuroinflammation and α-synuclein oligomerization by RWE may contribute to the improvement of PD pathology.