A comparison of reoperation rates after single-level anterior cervical discectomy and fusion (ACDF) between the procedures with and without anterior plate fixation.

INTRODUCTION: The plate fixation for anterior cervical discectomy and fusion (ACDF) has become increasingly widespread for facilitating early mobilization and improving fusion rate. However, apart from multilevel operations, there is still some controversy over its use for single-level ACDF. This retrospective study has compared the reoperation rates after single-level ACDFs performed at our institution between the procedures with and without plate fixation. METHODS: This retrospective study included a total of 131 patients with???1-year of follow-up after a single-level ACDF, consisting of 100 patients without plating (conventional ACDF) and 31 patients with plate fixation (plated ACDF). RESULTS: Eleven patients (8.4% of all patients):four conventional ACDFs (4% of the conventional ACDFs) and seven plated ACDFs (22.6% of the plated ACDFs), had reoperation surgeries. The incidence of reoperation was significantly higher in the plated ACDFs than in the conventional ACDFs (P=0.0037). The log-rank test revealed a significant difference (P=0.00003) in 5-year reoperation-free survival rates between the conventional (96.9%) and the plated groups (68.3%). CONCLUSION: Anterior cervical plating may have a negative impact on the adjacent segment integrity, resulting in an increased reoperation rate after a single-level ACDF at relatively shorter postoperative time points. J. Med. Invest. 70 : 334-342, August, 2023.

Refractory cardiac arrest caused by type I Kounis syndrome treated with adrenaline and nicorandil: A case report.

RATIONALE: Kounis syndrome is a rare but life-threatening anaphylactic reaction that can lead to acute coronary syndrome and cardiac arrest, and requires prompt diagnosis. Adrenaline, which is used to treat anaphylaxis, may cause coronary vasoconstriction and worsen ischemia, whereas coronary vasodilators may dilate systemic vessels and exacerbate hypotension. Delayed diagnosis of Kounis syndrome and inadequate therapeutic intervention may thus lead to a poor outcome. PATIENT CONCERNS: A 59-year-old man was treated for sepsis due to a liver abscess. Following administration of daptomycin, the patient developed severe anaphylactic shock leading to refractory cardiac arrest. Because conventional cardiopulmonary resuscitation was ineffective, extracorporeal cardiopulmonary resuscitation was considered as an alternative approach. DIAGNOSES: On bedside monitoring during cardiopulmonary resuscitation, unexpected ST-segment elevation was found on lead II electrocardiogram. Accordingly, the patient was clinically diagnosed with Kounis syndrome. INTERVENTIONS: Nicorandil (6 mg/h), a coronary vasodilator with minimal blood pressure effects, was administered along with high doses of vasopressors, including adrenaline 0.2 µg/kg/min. OUTCOMES: After the initiation of nicorandil administration, the patient achieved return of spontaneous circulation and did not require extracorporeal cardiopulmonary resuscitation. Based on the elevated serum tryptase level, normal creatine kinase-MB range, and lack of stenosis on coronary angiography, the patient was definitively diagnosed with type I (coronary vasospasm) Kounis syndrome. He was subsequently transferred to the referring hospital without neurological sequelae. LESSONS: If anaphylaxis leads to refractory shock and cardiac arrest, ischemic changes on the electrocardiogram should be investigated to identify underlying Kounis syndrome. In addition to adrenaline, coronary dilators are the definitive treatment. Nicorandil may be a useful treatment option because of its minimal effect on blood pressure.

Ternifolipyrons A-J: new cytotoxic α-pyrones from Isodon ternifolius (D. Don) Kudô.

Isodon ternifolius (D.Don) Kudô is an important Asian herb used in traditional medicine against several diseases. Nineteen compounds were isolated from the dichloromethane-methanol (1 : 1) extract of I. ternifolius roots, including ten new α-pyrone derivatives, named ternifolipyrons A-J. The chemical structures of the isolates were determined by a combination of 1D and 2D NMR, along with LR- and HRMS spectroscopy. The absolute configurations of the α-pyrone derivatives were constructed based upon the X-ray signal crystal of the bromobenzoyl derivative of 1 as well as the electronic circular dichroism (ECD). All isolates (1-19) were investigated for their growth-inhibitory potential towards CCRF-CEM-leukemia cells at a fixed concentration of 30 µM. The compounds which exerted more than 50% inhibition at this concentration, compounds (7, 10, 12, 15-17), were tested at a different concentration range to determine their IC50 values in CCRF-CEM leukemia, MDA-MB-231 triple-negative breast cancer, and MCF7 breast cancer cell lines. Ursolic acid (16) showed the most potent activity against the three cancer cell lines with IC50 values of 8.37, 18.04, and 18.93 µM, respectively.

Associations of serum leptin levels with intra-articular inflammatory cytokine levels in acute arthritic and nonarthritic knees of mice.

BACKGROUND: The roles of serum leptin in knee joint inflammation are unclear. The objective of this study was to identify any associations of serum leptin level with intra-articular inflammatory cytokine levels in acute arthritic and nonarthritic knees of mice. METHODS: Acute arthritis was induced by intra-articular injection of 2% carrageenan. Three groups (leptin-deficient ob/ob, wild-type (WT) and high-fat diet (HFD)-fed WT) were made. Serum leptin and inflammatory cytokines in the infrapatellar fat pad and synovium were measured before and 24 hr after injection. Affected knee joints were excised for histology 24 hr after injection. RESULTS: The HFD-WT group had significantly higher serum leptin than the ob/ob and WT groups before and after carrageenan injection. The HFD-WT group had significantly higher IL-1? and IL-6 in the infrapatellar fat pad and synovium than ob/ob and WT before injection but significantly lower IL-1?, IL-6 and TNF-? than the ob/ob group at 24 hr. CONCLUSIONS: Hyperleptinemia induced by a HFD is involved in low-grade intra-articular inflammation in nonarthritic knee joints. In contrast, leptin deficiency causes excessive intra-articular inflammation in carrageenan-induced acute arthritis. Leptin alleviates acute arthritis, while chronic hyperleptinemia is involved in low-grade inflammation in normal knee joints. J. Med. Invest. 70 : 54-59, February, 2023.

Antimicrobial metabolite of Cordyceps tenuipes targeting MurE ligase and histidine kinase via in silico study.

Cordyceps spp. are widely healthy foods around the world with several traditional uses and bio-functionalities. The chemical characterization of ethyl acetate-soluble extract of the entomopathogenic fungus Cordyceps tenuipes NBRC 111,630 afforded two new metabolites with 1,6-dioxaspiro[4.4]nonane motif, tenuipesone A (1) and tenuipesone B (2), along with four well-known metabolites (3-6). The elucidation of the chemical structures was carried out via extensive spectroscopic experiments including FTIR, HRMS, 1D-NMR, and 2D-NMR. The probable biosynthetic pathway of 1 and 2 was hypothesized. From the six isolates, beauvericin (6) exhibited antimicrobial activity against Bacillus subtilis and Staphylococcus aureus with respective MIC of 6.25 and 12.5 µM. Docking results exhibited that beauvericin (6) has significant binding affinities against MurE and HK proteins with ΔG = - 8.021 and - 8.585 kcal/mol, respectively. KEY POINTS: • Six compounds, including two new, were isolated from the entomopathogenic fungus Cordyceps tenuipes. • Plausible biosynthetic pathway of compounds 1, 2, 4, and 5 was hypothesized. • Beauvericin (6) exhibited significant antimicrobial activity against Bacillus subtilis and Staphylococcus aureus alongside binding affinities against MurE and HK proteins in MOE study.

A Novel Rat Model to Study Postsurgical Pain After Joint Replacement Surgery.

Purpose: The mechanisms underlying chronic postsurgical pain after joint replacement (JR) are complex, and it has been suggested that chronic postsurgical pain can develop as a result of inadequate acute pain management. Few studies have addressed acute pain after JR using specific animal models. This study aimed to develop a novel JR model focused on postsurgical pain assessment and the time course of pain recovery. Materials and Methods: Rats were allocated to the following three groups: sham (joint exposure), joint destruction (JD; resection of the femoral head), and JR (femoral head replacement using an originally developed implant). The time course of postsurgical pain behavior was measured using a dynamic weight-bearing apparatus, along with radiological assessments. The expression of calcitonin gene-related peptide-immunoreactive (CGRP-IR) neurons in the dorsal root ganglion (DRG) was evaluated by immunohistochemistry on days 28 and 42. Results: The ratio of weight-bearing distribution in the JR group gradually recovered from day 14 and reached the same level as that in the sham group on day 42, which was significantly greater than that in the JD group after day 7 (p<0.05). Radiologically, no significant issues were found, except for transient central migration of the implant in the JR group. The percentage of CGRP-IR DRG neurons in the JR group was significantly lower than that in the JD group on day 28 (mean, 37.4 vs 58.1%, p<0.05) and day 42 (mean, 32.3 vs 50.0%, p<0.05). Conclusion: Our novel JR model presented acute postsurgical pain behavior that was successfully recovered to the baseline level at day 42 after surgery. Difference of the pain manifestation between the JR and JD groups could be supported by the expression of CGRP-IR in DRG neurons. This model is the first step toward understanding detailed mechanisms of post-JR pain.

Finger trembling improvement after surgery in Hirayama disease: a case report.

INTRODUCTION: Finger trembling is a characteristic physical finding in Hirayama disease. Although conservative treatment is recommended to stop disease progression, surgery is optional in some cases. However, the postoperative recovery of finger trembling is scarcely reported. CASE PRESENTATION: A 26-year-old Japanese female patient whose chief complaint was left finger trembling with active finger extension presented at our hospital. Hand weakness without muscle atrophy of the left arm was observed. MRI showed left-side oriented intramedullary signal change with concomitant cord atrophy at C4-5 and C5-6. The CT myelogram (CTM) on flexion showed anterior cord compression and anterior shift of posterior dura matter from C4 to C6. And CTM on extension showed the resolution of both findings. Electrophysiological studies showed active and chronic neuronal damage and preserved motor neuron pool of hand muscle. Since she had exhibited a gradual aggravation of symptoms over a period of 5 years, she underwent anterior cervical discectomy and fusion after careful assessment of both conservative and surgical treatment. Finger trembling recovered soon after surgery. DISCUSSION: Finger trembling is an unfamiliar physical finding in terms of postoperative recovery prediction. Anterior horn cell impairment is postulated as a cause of finger trembling. Postural restoration of spinal cord shape and cerebrospinal fluid around the cord with preserved neural function could facilitate functional recovery.

Increased Calcitonin Gene-Related Peptide Expression in DRG and Nerve Fibers Proliferation Caused by Nonunion Fracture in Rats.

PURPOSE: Nonunion bone fracture can be a cause of persistent pain, but the pathophysiology remains largely unknown. The objective of this study was to identify how nonunion affect persistent pain after fracture. Specifically, we evaluated the association of neuropeptide change in dorsal root ganglia (DRG) and nerve proliferation at fracture sites with pain. METHODS: Rat union and nonunion fracture models were created. A piece of latex glove was placed at the fracture site to create a nonunion model. At 6 weeks after surgery, bone healing was assessed using radiography. In addition, the presence of calcitonin gene-related peptide-immunoreactive (CGRP-IR) DRG at the level of L3 and anti-growth associated protein 43-immunoreactive (GAP43-IR) nerve fibers in the scar tissue between the bone fragments were evaluated. Pain-related behavior was assessed using forced treadmill running. RESULTS: In radiological images at 6 weeks after surgery, callus formation was formed continuously between bone fragments in the union models. On the one hand, a clear gap was detected between fragments in nonunion models. The percentage of CGRP-IR DRG cells and the density of GAP43-IR nerve fibers in the scar tissue between the bone fragments in nonunion models was significantly higher than that in union models (p < 0.05). An increase in inflammatory cell infiltrate was observed in scar tissues in the nonunion models. During forced treadmill running, rats in the union model could run significantly longer than those in the nonunion models. CONCLUSION: Increased CGRP expression in DRG cells and abnormal nerve proliferation secondary to prolonged inflammation could lead to persistent pain after bone fracture. In clinical practice, early achievement of bone union may minimize the development of persistent pain after fractures.

A novel mice model of acute flares in osteoarthritis elicited by intra-articular injection of cultured mast cells.

PURPOSE: Mast cells are multifunctional in osteoarthritis (OA), and infiltration of activated mast cells likely contributes to disease severity and progression. However, the detailed mechanisms of action are unclear. The purpose of this study was to elucidate the role of mast cell infiltration in OA at histological level using a new mice model and to investigate pharmacological inhibitory effects of existing mast cell stabilizers in this model. METHODS: Mice were injected intra-articularly with monosodium iodoacetate (MIA 0.5 mg) or PBS on day 0, and PBS, with or without mast cells (MC: 1 × 106 cells) on day 14. They were divided into four groups: OA flare (MIA + MC), OA (MIA + PBS), MC non-OA (PBS + MC), and PBS non-OA (PBS + PBS). In OA flare, the MC stabilizer drug (tranilast: 400 mg/kg/day) or PBS was administered intraperitoneally from days 15 to 21. RESULTS: Histologically, modified Mankin score of the OA flare was significantly higher than that of OA (7.0 [1.8] vs. 3.3 [1.3], P < 0.05), and a larger number of mast cells was observed in OA flare than in OA (34.5 [6.3]/mm2 vs. 27.2 [2.3]/mm2, P < 0.05) on day 22. OA flare also showed acute exacerbation of pain and increased gene expression of pro-inflammatory cytokines and aggrecanase compared with OA. Administration of tranilast to OA flare-up provoked significant improvements in term of histological changes, pain, and gene expression at day 22. CONCLUSION: Our novel model possibly mimics OA flare conditions, which may open a new strategy of disease-modifying treatment for OA, focused on controlling the multiple functions of mast cells.

Design and synthesis of dual active neovibsanin derivatives based on a chemical structure merging method.

A hybrid compound consisting of neovibsanin and trans-banglene was designed according to a structure merging method and synthesized via a sequence of key steps including a Diels-Alder cycloaddition, stereoselective alkynylation, and intramolecular oxa-Michael addition reaction. The biological activity of the synthetized acetal compound and its hemiacetal analogue was investigated in PC12 cells. These studies revealed that the designed hybrid compounds displayed neuritogenic activity. Furthermore, a relatively strong neurite outgrowth promoting activity was observed in the presence of NGF. These results suggest that the designed hybrid compound exhibited a dual activity.

Raman image-activated cell sorting.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

Intelligent image-activated cell sorting 2.0.

The advent of intelligent image-activated cell sorting (iIACS) has enabled high-throughput intelligent image-based sorting of single live cells from heterogeneous populations. iIACS is an on-chip microfluidic technology that builds on a seamless integration of a high-throughput fluorescence microscope, cell focuser, cell sorter, and deep neural network on a hybrid software-hardware data management architecture, thereby providing the combined merits of optical microscopy, fluorescence-activated cell sorting (FACS), and deep learning. Here we report an iIACS machine that far surpasses the state-of-the-art iIACS machine in system performance in order to expand the range of applications and discoveries enabled by the technology. Specifically, it provides a high throughput of ∼2000 events per second and a high sensitivity of ∼50 molecules of equivalent soluble fluorophores (MESFs), both of which are 20 times superior to those achieved in previous reports. This is made possible by employing (i) an image-sensor-based optomechanical flow imaging method known as virtual-freezing fluorescence imaging and (ii) a real-time intelligent image processor on an 8-PC server equipped with 8 multi-core CPUs and GPUs for intelligent decision-making, in order to significantly boost the imaging performance and computational power of the iIACS machine. We characterize the iIACS machine with fluorescent particles and various cell types and show that the performance of the iIACS machine is close to its achievable design specification. Equipped with the improved capabilities, this new generation of the iIACS technology holds promise for diverse applications in immunology, microbiology, stem cell biology, cancer biology, pathology, and synthetic biology.

New alkaloidal metabolites from cultures of entomopathogenic fungus Cordyceps takaomontana NBRC 101754.

Two new alkaloidal metabolites, cordytakaoamides A (1) and B (2), as well as, 2-[(2-hydroxyethyl) amino] benzoic acid (3) and 2E-decenamide (4), and three known compounds (5-7) were isolated from ethyl acetate and n-butanol soluble portions of the entomopathogenic fungus, Cordyceps takaomontana NBRC 101754. Compounds 3 and 4 were isolated here for first time from natural resources. The chemical structures were established depending upon spectroscopic techniques such as 1D, 2D NMR, and HRMS. The absolute configuration of 1 and 2 was elucidated via the total synthesis of 1 as well as the experimental circular dichroism. Compound 3 was confirmed by a signal crystal X-ray analysis.

Author Correction: A practical guide to intelligent image-activated cell sorting.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-speed microparticle isolation unlimited by Poisson statistics.

High-speed isolation of microparticles (e.g., microplastics, heavy metal particles, microbes, cells) from heterogeneous populations is the key element of high-throughput sorting instruments for chemical, biological, industrial and medical applications. Unfortunately, the performance of continuous microparticle isolation or so-called sorting is fundamentally limited by the trade-off between throughput, purity, and yield. For example, at a given throughput, high-purity sorting needs to sacrifice yield, or vice versa. This is due to Poisson statistics of events (i.e., microparticles, microparticle clusters, microparticle debris) in which the interval between successive events is stochastic and can be very short. Here we demonstrate an on-chip microparticle sorter with an ultrashort switching window in both time (10 µs) and space (10 µm) at a high flow speed of 1 m s-1, thereby overcoming the Poisson trade-off. This is made possible by using femtosecond laser pulses that can produce highly localized transient cavitation bubbles in a microchannel to kick target microparticles from an acoustically focused, densely aligned, bumper-to-bumper stream of microparticles. Our method is important for rare-microparticle sorting applications where both high purity and high yield are required to avoid missing rare microparticles.

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

A practical guide to intelligent image-activated cell sorting.

Intelligent image-activated cell sorting (iIACS) is a machine-intelligence technology that performs real-time intelligent image-based sorting of single cells with high throughput. iIACS extends beyond the capabilities of fluorescence-activated cell sorting (FACS) from fluorescence intensity profiles of cells to multidimensional images, thereby enabling high-content sorting of cells or cell clusters with unique spatial chemical and morphological traits. Therefore, iIACS serves as an integral part of holistic single-cell analysis by enabling direct links between population-level analysis (flow cytometry), cell-level analysis (microscopy), and gene-level analysis (sequencing). Specifically, iIACS is based on a seamless integration of high-throughput cell microscopy (e.g., multicolor fluorescence imaging, bright-field imaging), cell focusing, cell sorting, and deep learning on a hybrid software-hardware data management infrastructure, enabling real-time automated operation for data acquisition, data processing, intelligent decision making, and actuation. Here, we provide a practical guide to iIACS that describes how to design, build, characterize, and use an iIACS machine. The guide includes the consideration of several important design parameters, such as throughput, sensitivity, dynamic range, image quality, sort purity, and sort yield; the development and integration of optical, microfluidic, electrical, computational, and mechanical components; and the characterization and practical usage of the integrated system. Assuming that all components are readily available, a team of several researchers experienced in optics, electronics, digital signal processing, microfluidics, mechatronics, and flow cytometry can complete this protocol in ~3 months.

Discrepancy between functional recovery and cutaneous silent period change in surgically treated degenerative cervical myelopathy: a prospective pilot study.

STUDY DESIGN: Exploratory research OBJECTIVES: Cutaneous silent periods (CSPs) that reflect the inhibitory spinal cord reflex, can sensitively detect spinal cord dysfunction, and contribute to the diagnosis of degenerative cervical myelopathy (DCM). However, CSP changes after DCM surgery related to functional improvement have not been reported. SETTING: University hospital in Nankoku, Japan METHODS: CSP recorded at four time points-before surgery, 3, 6, 12 months after surgery-were investigated in 31 hands of 16 DCM patients. CSPs were categorized as follows: normal, delayed onset latency, shortened duration, onset delay with shortened duration, and absent CSP. Myelopathic symptoms were evaluated by the Japanese Orthopaedic Association score (JOA score). RESULTS: Normal CSPs were observed in five hands (16%) before surgery and six hands (19%) twelve months after surgery (P > 0.05). Either onset delay or shortened duration or both were observed in 18 hands (58%) before surgery and 16 hands (52%) twelve months after surgery (P > 0.05). Absent CSPs were observed in eight hands (26%) before surgery and nine hands (29%) twelve months after surgery (P > 0.05). Measured values of onset latency and duration also did not change throughout the study period (P > 0.05). On the other hand, JOA scores improved after surgery. (P = 0.003). CONCLUSIONS: CSP abnormalities persisted after surgery in most cases, indicating irreversible damage of the intramedullary reflex circuit. JOA score recovery without CSP recovery provides insight into postoperative neural recovery in DCM.

High-throughput label-free molecular fingerprinting flow cytometry.

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm-1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.

Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.