Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.

Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.

Seizure-induced neutrophil adhesion in brain capillaries leads to a decrease in postictal cerebral blood flow.

Cerebral hypoperfusion has been proposed as a potential cause of postictal neurological dysfunction in epilepsy, but its underlying mechanism is still unclear. We show that a 30% reduction in postictal cerebral blood flow (CBF) has two contributing factors: the early hypoperfusion up to ∼30 min post-seizure was mainly induced by arteriolar constriction, while the hypoperfusion that persisted for over an hour was due to increased capillary stalling induced by neutrophil adhesion to brain capillaries, decreased red blood cell (RBC) flow accompanied by constriction of capillaries and venules, and elevated intercellular adhesion molecule-1 (ICAM-1) expression. Administration of antibodies against the neutrophil marker Ly6G and against LFA-1, which mediates adhesive interactions with ICAM-1, prevented neutrophil adhesion and recovered the prolonged CBF reductions to control levels. Our findings provide evidence that seizure-induced neutrophil adhesion to cerebral microvessels via ICAM-1 leads to prolonged postictal hypoperfusion, which may underlie neurological dysfunction in epilepsy.

Deep brain stimulation of the anterior nuclei of the thalamus can alleviate seizure severity and induce hippocampal GABAergic neuronal changes in a pilocarpine-induced epileptic mouse brain.

Deep brain stimulation (DBS) of the anterior nucleus of the thalamus (ANT) has been widely used as an effective treatment for refractory temporal lobe epilepsy. Despite its promising clinical outcome, the exact mechanism of how ANT-DBS alleviates seizure severity has not been fully understood, especially at the cellular level. To assess effects of DBS, the present study examined electroencephalography (EEG) signals and locomotor behavior changes and conducted immunohistochemical analyses to examine changes in neuronal activity, number of neurons, and neurogenesis of inhibitory neurons in different hippocampal subregions. ANT-DBS alleviated seizure activity, abnormal locomotor behaviors, reduced theta-band, increased gamma-band EEG power in the interictal state, and increased the number of neurons in the dentate gyrus (DG). The number of parvalbumin- and somatostatin-expressing inhibitory neurons was recovered to the level in DG and CA1 of naïve mice. Notably, BrdU-positive inhibitory neurons were increased. In conclusion, ANT-DBS not only could reduce the number of seizures, but also could induce neuronal changes in the hippocampus, which is a key region involved in chronic epileptogenesis. Importantly, our results suggest that ANT-DBS may lead to hippocampal subregion-specific cellular recovery of GABAergic inhibitory neurons.

Ventricular tachycardia of right ventricular outflow tract origin during the perioperative period: A case report.

RATIONALE: Idiopathic ventricular tachycardia (VT) occurs in individuals without structural abnormalities in the heart, accounts for approximately 10% of total VTs. Furthermore, approximately 70% of idiopathic VTs originate from Right ventricular outflow tract (RVOT). However, among perioperative arrhythmias, incidence of VT after surgery is extremely rare and most arrhythmias are atrial origin. PATIENT CONCERNS: A 69-year-old man with permanent pacemaker underwent colon surgery. DIAGNOSES: Patient suffered from low blood pressure and dizziness, sweating at post anesthetic care unit (PACU) and heart rate (HR) increased suddenly to 200 beats/min with monomorphic VT after bolus ephedrine administration and continuous dopamine infusion. INTERVENTIONS: Pacemaker interrogation followed by DC cardioversion was done. OUTCOMES: Patient's vital signs became normal and symptoms are subsided. LESSONS: RVOT VT can be caused by triggering activities, such as ephedrine, dopamine, and inadequate fluid management. These triggering activities are initiated by acceleration of HR from ventricles with infusion of catecholamine which lead monomorphic VT originating from RVOT.RVOT origin PVCs can be precipitated into monomorphic VT by administrating catecholamines such as ephedrine and dopamine even in patient with pacemaker. The mechanism of these VTs includes catecholamine induced acceleration of HR. Since RVOT PVCs can be recognize by 12 EKGs, we should be pay more attentions to the pre-operation EKG and be cautious using catecholamines.

Differential contribution of excitatory and inhibitory neurons in shaping neurovascular coupling in different epileptic neural states.

Understanding the neurovascular coupling (NVC) underlying hemodynamic changes in epilepsy is crucial to properly interpreting functional brain imaging signals associated with epileptic events. However, how excitatory and inhibitory neurons affect vascular responses in different epileptic states remains unknown. We conducted real-time in vivo measurements of cerebral blood flow (CBF), vessel diameter, and excitatory and inhibitory neuronal calcium signals during recurrent focal seizures. During preictal states, decreases in CBF and arteriole diameter were closely related to decreased γ-band local field potential (LFP) power, which was linked to relatively elevated excitatory and reduced inhibitory neuronal activity levels. Notably, this preictal condition was followed by a strengthened ictal event. In particular, the preictal inhibitory activity level was positively correlated with coherent oscillating activity specific to inhibitory neurons. In contrast, ictal states were characterized by elevated synchrony in excitatory neurons. Given these findings, we suggest that excitatory and inhibitory neurons differentially contribute to shaping the ictal and preictal neural states, respectively. Moreover, the preictal vascular activity, alongside with the γ-band, may reflect the relative levels of excitatory and inhibitory neuronal activity, and upcoming ictal activity. Our findings provide useful insights into how perfusion signals of different epileptic states are related in terms of NVC.

Abdominal compartment syndrome caused by gastric distension in bulimia nervosa and fatal injury following surgical decompression - A case report.

BACKGROUND: Abdominal compartment syndrome (ACS) occurs due to increased abdominal cavity pressure, causes multiple organ damage, and leads to fatal consequences. Increased intraperitoneal pressure due to different reasons generally does not result in serious damage, due to the compliance of the abdominal wall. However, when the pressure exceeds the limit, ACS develops, thereby causing fatal damage to the organs. CASE: A patient presented with rapid stomach swelling due to excessive food intake and was known to have bulimia nervosa, which had now resulted in ACS. Mental changes, abdominal distension, color change in the legs, acute kidney injury, and acidosis were seen. The patient expired due to ischemia-reperfusion injury and disseminated intravascular coagulation, which occurred after surgical decompression. CONCLUSIONS: Under suspected ACS conditions, we should be aware of various symptoms that can occur. Early attempts for decompression are helpful, and it is important to be prepared for reperfusion injury prior to surgical decompression attempts.

Excitation-Inhibition Imbalance Leads to Alteration of Neuronal Coherence and Neurovascular Coupling under Acute Stress.

A single stressful event can cause morphologic and functional changes in neurons and even malfunction of vascular systems, which can lead to acute stress disorder or post-traumatic stress disorder. However, there is a lack of evidence regarding how acute stress impacts neuronal activity, the concurrent vascular response, and the relationship between these two factors, which is defined as neurovascular coupling. Here, using in vivo two-photon imaging, we found that NMDA-evoked calcium transients of excitatory neurons were impaired and that vasodilation of penetrating arterioles was concomitantly disrupted in acutely stressed male mice. Furthermore, acute stress altered the relationship between excitatory neuronal calcium coherence and vascular responses. By measuring NMDA-evoked excitatory and inhibitory neuronal calcium activity in acute brain slices, we confirmed that neuronal coherence both between excitatory neurons and between excitatory and inhibitory neurons was reduced by acute stress but restored by blockade of glucocorticoid receptor signaling. Furthermore, the ratio of sEPSCs to sIPSCs was altered by acute stress, suggesting that the excitation-inhibition balance was disrupted by acute stress. In summary, in vivo, ex vivo, and whole-cell recording studies demonstrate that acute stress modifies excitatory-inhibitory neuronal coherence, disrupts the excitation-inhibition balance, and causes consequent neurovascular coupling changes, providing critical insights into the neural mechanism of stress-induced disorders.SIGNIFICANCE STATEMENT Acute stress can cause pathologic conditions, such as acute stress disorder and post-traumatic stress disorder, by affecting the functions of neurons and blood vessels. However, investigations into the impacts of acute stress on neurovascular coupling, the tight connection between local neural activity and subsequent blood flow changes, are lacking. Through investigations at the in vivo, ex vivo, and whole-cell recording levels, we found that acute stress alters the NMDA-evoked vascular response, impairs the function and coherence of excitatory and inhibitory neurons, and disrupts the excitatory and inhibitory balance. These novel findings provide insights into the relevance of the excitatory-inhibitory balance, neuronal coherence, and neurovascular coupling to stress-induced disorders.

Supramolecular Peptide Hydrogel-Based Soft Neural Interface Augments Brain Signals through a Three-Dimensional Electrical Network.

Recording neural activity from the living brain is of great interest in neuroscience for interpreting cognitive processing or neurological disorders. Despite recent advances in neural technologies, development of a soft neural interface that integrates with neural tissues, increases recording sensitivity, and prevents signal dissipation still remains a major challenge. Here, we introduce a biocompatible, conductive, and biostable neural interface, a supramolecular ß-peptide-based hydrogel that allows signal amplification via tight neural/hydrogel contact without neuroinflammation. The non-biodegradable ß-peptide forms a multihierarchical structure with conductive nanomaterial, creating a three-dimensional electrical network, which can augment brain signal efficiently. By achieving seamless integration in brain tissue with increased contact area and tight neural tissue coupling, the epidural and intracortical neural signals recorded with the hydrogel were augmented, especially in the high frequency range. Overall, our tissuelike chronic neural interface will facilitate a deeper understanding of brain oscillation in broad brain states and further lead to more efficient brain-computer interfaces.

A comparison of palonosetron and dexamethasone for postoperative nausea and vomiting in orthopedic patients receiving patient-controlled epidural analgesia.

BACKGROUND: Postoperative nausea and vomiting (PONV) is one of the major concerns after anesthesia and surgery, and it may be more frequent in orthopedic patients receiving patient-controlled epidural analgesia (PCEA). The purpose of this study was to compare the effect of palonosetron and dexamethasone on the prevention of PONV in patients undergoing total joint arthroplasty and receiving PCEA. METHODS: Patients scheduled for total hip or knee arthroplasty under spinal anesthesia/PCEA were randomly allocated to receive either intravenous palonosetron (0.075 mg, n = 50) or dexamethasone (5 mg, n = 50). Treatments were administered intravenously to the patients 30 min before the beginning of surgery. The total incidence of PONV and incidence in each time period, severity of nausea, need for rescue anti-emetics, pain score, and adverse effects during the first 48 h postoperatively were evaluated. RESULTS: The total incidence of PONV was lower in the palonosetron group compared with the dexamethasone group (18.4% vs. 36.7%, P = 0.042), but there were no statistically significant differences in incidence between the groups at all time points. No significant intergroup differences were observed in the severity of nausea, use of rescue anti-emetics, pain score, and adverse effects. CONCLUSIONS: Although there were no significant differences in the incidence of PONV between the treatment groups at all time points, intravenous palonosetron reduced the total incidence of PONV in orthopedic patients receiving PCEA compared with dexamethasone.

Relationship between first dorsal sacral foramen and lumbar facet joint connecting line in South Korea populations: Retrospective study.

The first sacral nerve root block (S1 nerve root block) is a practical procedure for patients with radiating lower back pain. In general, S1 nerve root block is performed under x-ray fluoroscopy. It is necessary to adjust the position of the patient and angle of fluoroscopy to properly visualize the first dorsal sacral foramen (dorsal S1 foramen). The purpose of this study was to analyze the location of dorsal S1 foramen and lumbar facet joint in S1 nerve root block.A total of 388 patients undergoing x-ray fluoroscopy-guided S1 nerve root block in the prone position were examined. X-ray fluoroscopy was fixed at the corresponding location of facet joint of L4-5 and L5-S1. The relationship of the connecting line vertical to L5-S1 facet joint and upper margin sacrum was evaluated. The surface anatomical relationships between dorsal S1 foramen and lumbar facet joint were assessed.Based on the localization of dorsal S1 foramen, the line drawn from point to upper margin sacrum passed through the dorsal S1 foramen in all cases. The horizontal distance from the spinous process to the dorsal S1 foramen was 25.9 ±â€Š3.0 mm for men and 26.2 ±â€Š1.4 mm for women. The horizontal distance from the between posterior superioriliac spine to the dorsal S1 foramen was 26.2 ±â€Š2.7 mm for men and 26.8 ±â€Š1.7 mm for women. The vertical distance from the upper margin of sacrum to dorsal S1 foramen to the dorsal S1 foramen was 45.6 ±â€Š6.5 mm for men and 43.8 ±â€Š6.0 mm for women.The connecting line vertical to L5-S1 facet joint and upper margin sacrum is located on the same line from the dorsal S1 foramen. For difficult cases of locating dorsal S1 foramen, the lumbar facet joint can assist in predicting the vertical location of the dorsal S1 foramen.

Segregation for reduction of regulated medical waste in the operating room: a case report.

One-third of all hospital-regulated medical waste (RMW) comes from the operating room (OR), and it considerably consists of disposable packaging and wrapping materials for the sterilization of surgical instruments. This study sought to identify the amount and type of waste produced by ORs in order to reduce the RMW so as to achieve environmentally-friendly waste management in the OR. We performed an initial waste segregation of 4 total knee replacement arthroplasties (TKRAs) and 1 total hip replacement arthroplasty, and later of 1 extra TKRA, 1 laparoscopic anterior resection of the colon, and 1 pelviscopy (with radical vaginal hysterectomy), performed at our OR. The total mass of non-regulated medical waste (non-RMW) and blue wrap amounted to 30.5 kg (24.9%), and that of RMW to 92.1 kg (75.1%). In the course of the study, we noted that the non-RMW included recyclables, such as papers, plastics, cardboards, and various wrapping materials. The study showed that a reduction in RMW generation can be achieved through the systematic segregation of OR waste.

Surface Landmarks do not Correspond to Exact Levels of the Cervical Spine: References According to the Sex, Age and Height.

OBJECTIVE: A general orientation along the cervical spine could be estimated by external landmarks, and it was useful, quick and less exposable to radiation, but, sometimes it gave reference confusion of target cervical level. The authors reviewed the corresponding between the neck external landmarks and cervical levels. METHODS: Totally 1,031 cervical lateral radiographs of different patients were reviewed in single university hospital. Its compositions were 534 of males and 497 females; 86 of second decades (10-19 years-old), 169 of third decades, 159 of fourth decades, 209 of fifth decades, 275 of sixth decades, and 133 of more than seventh decades (>60 years-old). Reference external landmarks (mandible, hyoid bone, thyroid cartilage, and cricothyroid membrane) with compounding factors were reviewed. RESULTS: The reference levels of cervical landmarks were C2.13 with mandible angle, C3.54 with hyoid bone, C5.12 with thyroid cartilage, and C6.01 with cricothyroid membrane. The reference levels of cervical landmarks were differently observed by sex, age, and somatometric measurement (height) accordingly mandible angle from C1 to C3, hyoid bone from disc level of C2 and C3 to C5, thyroid cartilage from disc level of C3 and C4 to C7, and cricothyroid membrane from C4 to disc level of C7 and T1. CONCLUSION: Surface landmarks only provide general reference points, but not correspond to exact levels of the cervical spine. Intraoperative fluoroscopy ensures a more precise placement to the targeted cervical level.

Asymptomatic pneumomediastinum resulting from air in the epidural space -a case report-.

There are no reports regarding pneumomediastinum caused by thoracic epidural block complications. We believe that it is possible to experience an occurrence of pneumomediastinum caused by air in the epidural space after performing a thoracic epidural block using the loss of resistance (LOR) technique with air. We report a witnessed case where pneumomediastinum appeared after a thoracic epidural block. Pneumorrrhachis, paravertebral muscle emphysema, and pneumomediastinum were diagnosed by Positron Emission Tomography-Computed Tomography. Although extremely rare, pneumomediastinum can be caused by an epidural block using LOR technique with air. In order to avoid the above danger, the use of saline or very minimal amount of air is required during a careful LOR technique.

Successful resuscitation of cardiac arrest caused by CO2 embolism with intra-aortic injection of epinephrine during off-pump coronary bypass surgery -a case report-.

Although compressed gas (CO2) blowers have been used safely to aid accurate grafting during off-pump coronary bypass surgery, hemodynamic collapse due to gas embolism into the right coronary artery may occur. Supportive measures to facilitate gas clearance by increasing the coronary perfusion pressure have been reported to be successful in restoring hemodynamic stability. However, right ventricular dysfunction and atrioventricular nodal ischemia may hinder effective systemic delivery of the vasoactive medications, even when performing resuscitative measures such as direct cardiac massage. We herein report a case of cardiac arrest that was caused by a right coronary gas embolism and that could not be restored by cardiac resuscitation. When supportive measures fail, direct aortic injection of epinephrine to increase the coronary perfusion pressure can be attempted before initiating cardiopulmonary bypass, and this approach may be life-saving in situations that limit systemic drug delivery from the venous side despite the performance of direct cardiac massage.

The psychopathological influence of congenital heart disease in Korean male adolescents: an analysis of multiphasic personal inventory test results.

PURPOSE: The aim of this study was to evaluate the psychopathological influence of congenital heart disease (CHD) in Korean 19-year-old males. MATERIALS AND METHODS: The authors compared the Korean military multiphasic personal inventory (KMPI) military profiles of 211 CHD cases (atrial septal defect, ventricular septal defect, patent ductus arteriosus, or combined CHD) with the KMPI profiles of 300 normal controls. The CHD group was also divided according to whether or not the subjects had undergone open cardiac surgery in order to evaluate the psychopathological effects of an operation among the subjects. RESULTS: A decreased result on the faking-good response scale and an increased result on the faking-bad response were observed in the CHD group compared to the control (p<0.01). The neurosis scale results, including anxiety, depression and somatization symptoms, were markedly increased in the CHD group compared to the control (p<0.01). The severity level of personality disorder was also increased in the CHD group (p<0.001). Differences in KMPI scale scores were not related to open cardiac surgery history. CONCLUSION: In this study, young males with CHD tended to report more abnormal results on the multiphasic personal inventory test in comparison to normal subjects, suggesting that CHD may be related to psychopathology in young males in Korea. Therefore, clinicians are recommended to evaluate the psychopathological traits of patients with CHD.

Minimal volume of local anesthetic required for an ultrasound-guided SGB.

BACKGROUND: Compared with the blind technique, ultrasound-guided stellate ganglion block (SGB) reduces the amount of local anesthetic needed for a successful block. The purpose of this study is to determine the minimal, optimal volume of local anesthetic required for successful ultrasound-guided SGB and to reduce its adverse effects. METHODS: Thirty-five patients with postherpetic neuralgia and complex regional pain syndrome of the upper extremity and the facial area were selected. For ultrasound-guided SGB by subfacial method, each patient was injected with 0.5% mepivacaine mixed with contrast media in 2 mL, 3 mL, and 4 mL doses at 2-week intervals. After the procedure, the spread of contrast media in the spine was checked by fluoroscopy. Ptosis and conjunctival flushing were rated and recorded. Adverse effects, such as hoarseness, foreign body sensation, swallowing difficulty, and upper arm weakness, were also recorded. RESULTS: Out of the 35 initial patients, the results for 33 patients who received all three SGBs were included in this study. The contrast media spread to 4.80 ± 0.82, 4.94 ± 0.86, and 5.09 ± 0.97 total spinal segments in the 2 mL, 3 mL, and 4 mL groups, respectively. The cephalad spread of contrast media was 2.16 ± 0.74, 2.23 ± 0.85, and 2.30 ± 0.78 spinal segments for the 2 mL, 3 mL, and 4 mL groups, respectively, and the caudad spread of contrast media was 2.64 ± 0.51, 2.70 ± 0.61, and 2.89 ± 0.64 segments in the 2 mL, 3 mL, and 4 mL groups, respectively. There were no significant statistical differences in any segments for the three groups (P > 0.05). Review of the fluoroscopic images showed spread of the contrast media below the C7-T1 junction in all three groups. Ptosis developed in all three groups after the procedure. CONCLUSION: In conclusion, when performing an ultrasound-guided SGB, 2 mL dosage was sufficient for a successful block as the previous, conventional volume. Therefore, when performing an ultrasound-guided SGB, we recommend the 2 mL dosage of local anesthetics for a successful block.

Effects of increasing the dose of ropivacaine on vertical infraclavicular block using neurostimulation.

BACKGROUND: Use of an infraclavicular block is appropriate for surgery of the upper limb. However, it does not consistently block the entire brachial plexus. The aim of this study was to investigate whether increasing the dose of ropivacaine could enhance the success rate, onset time, and efficacy of the sensory and motor block during the use of a vertical infraclavicular block using neurostimulation in upper limb surgery. METHODS: TWO HUNDREDS AND TEN PATIENTS WERE PROSPECTIVELY RANDOMIZED INTO THREE GROUPS: Group 1 (30 ml of 0.5% ropivacaine; n = 70), Group 2 (40 ml of 0.5% ropivacaine; n = 70), and Group 3 (40 ml of 0.75% ropivacaine; n = 70). Patients in each group received a vertical infraclavicular block using neurostimulation and obtained a distal motor response of the ulnar or median nerve. Recorded outcome measures included block success rate, onset time, sensory and motor blocks, and adverse events. RESULTS: No differences were found in the block success rate among the three groups (92.8%, 97.1%, and 94.2% for Groups 1, 2, and, 3, respectively; P = 0.346). There were no significant differences in onset time (P = 0.225) among groups, nor was there enhancement in the sensory block, but the motor block was enhanced. Local anesthetic toxicity was observed in five female patients from group 3 (P = 0.006). CONCLUSIONS: Although the efficacy of the motor block was significantly improved, success rate, onset time, and efficacy of sensory block were not enhanced significantly among groups despite differences in volume and volume/concentration of the local anesthetic.