Large-Scale Whole-Genome Analysis of HTLV-1-Associated Myelopathy Identified Hereditary Spastic Paraplegias.

Objectives: Distinguishing human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy from hereditary spastic paraplegia in patients infected with HTLV-1 is challenging due to overlapping clinical symptoms. The aim of this study was to explore the possibility that hereditary spastic paraplegia is inherently present in patients diagnosed with HTLV-1-associated myelopathy. Methods: We performed whole-genome sequencing on 315 unrelated patients registered in the HTLV-1-Associated Myelopathy patient registry "HAM-net," from 2013 to 2022 in Japan. CSF inflammatory biomarkers, including CXCL10, were measured. Results: We identified 5 patients with pathogenic variants in the genes RTN2, SPAST, VCP, and UBAP1, which are the known causes of hereditary spastic paraplegia. These patients had no family history of hereditary spastic paraplegia. The levels of CSF inflammatory biomarkers were lower than expected in these patients, compared with disease severity. Discussion: Genetic analysis is useful for the differentiation of hereditary spastic paraplegia patients from HTLV-1-associated myelopathy patients, especially for the patients with low levels of CSF inflammatory markers. Here we report the presence of hereditary spinal cord diseases in patients diagnosed with HTLV-1-associated myelopathy and provides evidence that genetic analysis would be helpful in the diagnostic workflow.

Isolation and Structure Determination of New Pyrones from Dictyostelium spp. Cellular Slime Molds Coincubated with Pseudomonas spp.

Cellular slime molds are excellent model organisms in the field of cell and developmental biology because of their simple developmental patterns. During our studies on the identification of bioactive molecules from secondary metabolites of cellular slime molds toward the development of novel pharmaceuticals, we revealed the structural diversity of secondary metabolites. Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without using medium components. Although changing the feeding bacteria is expected to affect dramatically the secondary metabolite production, the effect of the feeding bacteria on the production of secondary metabolites is not known. Herein, we report the isolation and structure elucidation of clavapyrone (1) from Dictyostelium clavatum, intermedipyrone (2) from D. magnum, and magnumiol (3) from D. intermedium. These compounds are not obtained from usual cultural conditions with Klebsiella aerogenes but obtained from coincubated conditions with Pseudomonas spp. The results demonstrate the diversity of the secondary metabolites of cellular slime molds and suggest that widening the range of feeding bacteria for cellular slime molds would increase their application potential in drug discovery.

Quantitative assessment of anterior talofibular ligament quality in chronic lateral ankle instability using magnetic resonance imaging T2* value.

OBJECTIVE: To determine T2* normal reference values for anterior talofibular ligament (ATFL) and to investigate the feasibility of the quantitative ATFL quality evaluation in chronic lateral ankle instability (CLAI) using T2* values. MATERIALS AND METHODS: This study enrolled 15 patients with CLAI and 30 healthy volunteers. The entire ATFL T2* values from the MRI T2* mapping were measured. The prediction equation (variables: age, height, and weight) in a multiple linear regression model was used to calculate the T2* normal reference value in the healthy group. T2* ratio was defined as the ratio of the actual T2* value of the patient's ATFL to the normal reference value for each patient. A Telos device was used to measure the talar tilt angle (TTA) from the stress radiograph. RESULTS: T2* values of ATFL in the healthy and CLAI groups were 10.82 ± 1.84 ms and 14.36 ± 4.30 ms, respectively, which are significantly higher in the CLAI group (P < 0.05). The prediction equation of the normal reference T2* value was [14.9 + 0.14 × age (years) - 4.7 × height (m) - 0.03 × weight (kg)] (R2 = 0.65, P < 0.0001). A significant positive correlation was found between the T2* ratio and TTA (r = 0.66, P = 0.007). CONCLUSION: MRI T2* values in patients with CLAI were higher than those in healthy participants, and the T2* ratio correlated with TTA, suggesting that T2* values are promising for quantitative assessment of ATFL quality preoperatively.

Characteristics of Nonunion After Arthroscopic Ankle Arthrodesis: A Multicenter Retrospective Cohort Study.

Nonunion is a major complication of arthroscopic ankle arthrodesis. However, the characteristics and risk factors of nonunion are not well understood. This retrospective multicenter observational study aimed to clarify the characteristics of nonunion after arthroscopic ankle arthrodesis. We included 154 patients who underwent arthroscopic ankle arthrodesis at any 1 of 5 institutions. Patients were divided into 2 groups: union and nonunion, and the groups were compared. Age, sex, body mass index, diabetes, smoking, corticosteroid use, diagnosis, treatment information, treatment protocol, radiographic evaluation, and patient-reported outcomes were recorded and analyzed. On radiographs, bony union was observed in 142 ankles (91.0%) but not in 12 ankles (9.0%). Postoperative radiographic tibial bony gap (mm) was significantly larger in the nonunion group (medial = 1.98, center = 1.65, anterior = 2.21, middle = 1.72, posterior = 3.01) than in the union group (medial = 1.35, center = 1.13, anterior = 1.28, middle = 1.03, posterior = 2.03). Furthermore, the visual analog score (VAS) of pain and pain-related self-administered foot evaluation questionnaire (SAFE-Q) subscale score significantly worsened in the nonunion group (VAS = 3.83, SAFE-Q subscale score = 69.8) compared to that in the union group (VAS = 1.35, SAFE-Q subscale score = 76.6). A larger radiographic tibiotalar bony gap was observed in the nonunion group. Other measurement outcomes were not associated with nonunion. Additionally, patient-reported outcomes markedly worsened in the nonunion group.

The function of cruciate ligaments in bi-cruciate retaining Total knee arthroplasty with asymmetrical design.

BACKGROUND: Bi-cruciate retaining total knee arthroplasty with an asymmetrical design may improve knee function and clinical outcomes. This study aimed to compare the kinematics, anteroposterior laxity, and in situ forces of the anterior and posterior cruciate ligaments of knees subjected to this treatment with those of healthy knees. METHODS: Seven fresh-frozen cadaveric knees were tested using a robotic/universal force-moment sensor system. The kinematics during passive flexion-extension motion and anteroposterior laxity for native knee, treated knee, and treated knee with cruciate ligament transection states were investigated. The motions of the intact and treated knees during each test were repeated after anterior/posterior cruciate ligament transection to calculate the in situ force in the ligaments. FINDINGS: The screw-home movement of normal knees disappeared after treatment. The in situ force of the anterior cruciate ligament in treated knees was higher than that in intact knees at ˃15° during flexion and at 60° and 90° against an anterior force. The in situ force of the posterior cruciate ligament in treated knees was higher at 0°, 15°, and 30° during flexion and at all flexion angles against a posterior force. INTERPRETATION: The screw-home movement of normal knees decreased, and the in situ force of the anterior and posterior cruciate ligaments increased after treatment.

EZH1/2 dual inhibitors suppress HTLV-1-infected cell proliferation and hyperimmune response in HTLV-1-associated myelopathy.

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy (HAM), adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated uveitis, and pulmonary diseases. Although both HAM and ATL show proliferation of infected cells, their pathogeneses are quite different. In particular, the pathogenesis of HAM is characterized by hyperimmune responses to HTLV-1-infected cells. Recently, we demonstrated the overexpression of histone methyltransferase EZH2 in ATL cells and the cytotoxic effects of EZH2 inhibitors and EZH1/2 dual inhibitors on these cells. However, these phenomena have never been studied in HAM. Furthermore, what effect these agents have on the hyperimmune response seen in HAM is completely unknown. Methods: In this study, we investigated histone methyltransferase expression levels in infected cell populations (CD4+ and CD4+CCR4+ cells) from patients with HAM using microarray and RT-qPCR analyses. Next, using an assay system that utilizes the spontaneous proliferation characteristic of peripheral blood mononuclear cells derived from patients with HAM (HAM-PBMCs), we investigated the effects of EZH2 selective inhibitors (GSK126 and tazemetostat) and EZH1/2 dual inhibitors (OR-S1 and valemetostat, also known as DS-3201), particularly on cell proliferation rate, cytokine production, and HTLV-1 proviral load. We also examined the effect of EZH1/2 inhibitors on the proliferation of HTLV-1-infected cell lines (HCT-4 and HCT-5) derived from patients with HAM. Results: We found elevated expression of EZH2 in CD4+ and CD4+CCR4+ cells from patients with HAM. EZH2 selective inhibitors and EZH1/2 inhibitors significantly inhibited spontaneous proliferation of HAM-PBMC in a concentration-dependent manner. The effect was greater with EZH1/2 inhibitors. EZH1/2 inhibitors also reduced the frequencies of Ki67+ CD4+ T cells and Ki67+ CD8+ T cells. Furthermore, they reduced HTLV-1 proviral loads and increased IL-10 levels in culture supernatants but did not alter IFN-γ and TNF-α levels. These agents also caused a concentration-dependent inhibition of the proliferation of HTLV-1-infected cell lines derived from patients with HAM and increased annexin-V(+)7-aminoactinomycin D(-) early apoptotic cells. Conclusion: This study showed that EZH1/2 inhibitors suppress HTLV-1-infected cell proliferation through apoptosis and the hyperimmune response in HAM. This indicates that EZH1/2 inhibitors may be effective in treating HAM.

High reproducibility of a novel supported anterior drawer test for diagnosing ankle instability.

BACKGROUND: The manual traditional anterior drawer test (ADT) is essential for deciding the treatment for chronic ankle instability, but it has been shown to have a comparatively low reproducibility and accuracy, especially in less experienced hands. To clarify the inter-examiner reproducibility, we compared the actual distance of anterior translation between junior and senior examiners in ADT. We also evaluated the diagnostic abilities of traditional ADT, and a novel modified ADT (supported ADT). METHODS: Thirty ankles were included in this study, and ankle instability was defined using stress radiography. All subjects underwent two methods of manual ADT by junior and senior examiners, and ankle instability was judged in a blinded fashion. The anterior drawer distance was calculated from the lengthening measured using a capacitance-type sensor device. RESULTS: The degree of anterior translation determined by the junior examiner was significantly lower than that determined by the senior examiner when traditional ADT was performed (3.3 vs. 4.5 mm, P = 0.016), but there was no significant difference in anterior translation between the two examiners when supported ADT was performed (4.6 vs. 4.1 mm, P = 0.168). The inter-examiner reliability of supported ADT was higher than that of traditional ADT. For the junior examiner, the diagnostic accuracy of supported ADT was higher than that of traditional ADT (sensitivity, 0.40 vs. 0.80; specificity, 0.75 vs. 0.80). CONCLUSION: Supported ADT may have the advantage of being a simple manual test of ankle instability with less error between examiners.

The Role of the Medial Meniscus in Anterior Knee Stability.

Background: Few studies have compared the force distribution between the anterolateral, posterolateral, and medial structures of the knee. Purpose: To investigate the important structures in an intact knee contributing to force distribution in response to anterior tibial load. Study Design: Controlled laboratory study. Methods: Nine fresh-frozen cadaveric knee specimens underwent robotic testing. First, 100 N of anterior tibial load was applied to the intact knee at 0°, 15°, 30°, 60°, and 90° of knee flexion. The anterior cruciate ligament (ACL), anterolateral capsule, lateral collateral ligament, popliteal tendon, posterior root of the lateral meniscus, superficial medial collateral ligament, posterior root of the medial meniscus (MM), and posterior cruciate ligament were then completely transected in sequential order. After each transection, the authors reproduced the intact knee motion when a 100-N anterior tibial load was applied. By applying the principle of superposition, the resultant force of each structure was determined based on the 6 degrees of freedom force/torque data of each state. Results: At every measured knee flexion angle, the resultant force of the ACL was the largest of the tested structures. At knee flexion angles of 60° and 90°, the resultant force of the MM was larger than that of all other structures with the exception of the ACL. Conclusion: The MM was identified as playing an important role in response to anterior tibial load at 60° and 90° of flexion. Clinical Relevance: In clinical settings, the ACL of patients with a poorly functioning MM, such as tear of the MM posterior root, should be monitored considering the large resultant force in response to an anterior tibial load.

Potential role of HTLV-1 Tax-specific cytotoxic t lymphocytes expressing a unique t-cell receptor to promote inflammation of the central nervous system in myelopathy associated with HTLV-1.

Human T-lymphotropic virus 1 (HTLV-1) infection causes two serious diseases: adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy (HAM). Immunological studies have revealed that HTLV-1 Tax-specific CD8+ cytotoxic T-cells (Tax-CTLs) in asymptomatic carriers (ACs) and ATL patients play an important role in the elimination of HTLV-1-infected host cells, whereas Tax-CTLs in HAM patients trigger an excessive immune response against HTLV-1-infected host cells infiltrating the central nervous system (CNS), leading to local inflammation. Our previous evaluation of HTLV-1 Tax301-309 (SFHSLHLLF)-specific Tax-CTLs (Tax301-309-CTLs) revealed that a unique T-cell receptor (TCR) containing amino acid (AA)-sequence motif PDR, was shared among HLA-A*24:02+ ACs and ATL patients and behaved as an eliminator by strong activity against HTLV-1. However, it remains unclear whether PDR+Tax301-309-CTLs also exist in HLA-A*24:02+ HAM patients and are involved in the pathogenesis of HAM. In the present study, by high-throughput TCR repertoire analysis technology, we revealed TCR repertoires of Tax301-309-CTLs in peripheral blood (PB) of HLA-A*24:02+ HAM patients were skewed, and a unique TCR-motif PDR was conserved in HAM patients (10 of 11 cases). The remaining case dominantly expressed (-DR, P-R, and PD-), which differed by one AA from PDR. Overall, TCRs with unique AA-sequence motifs PDR, or (-DR, P-R, and PD-) accounted for a total of 0.3-98.1% of Tax301-309-CTLs repertoires of HLA-A*24:02+ HAM patients. Moreover, TCR repertoire analysis of T-cells in the cerebrospinal fluid (CSF) from four HAM patients demonstrated the possibility that PDR+Tax301-309-CTLs and (-DR, P-R, and PD-)+Tax301-309-CTLs efficiently migrated and accumulated in the CSF of HAM patients fostering increased inflammation, although we observed no clear significant correlation between the frequencies of them in PB and the levels of CSF neopterin, a known disease activity biomarker of HAM. Furthermore, to better understand the potential function of PDR+Tax301-309-CTLs, we performed immune profiling by single-cell RNA-sequencing of Tax301-309-CTLs, and the result showed that PDR+Tax301-309-CTLs up-regulated the gene expression of natural killer cell marker KLRB1 (CD161), which may be associated with T-cell activation and highly cytotoxic potential of memory T-cells. These findings indicated that unique and shared PDR+Tax301-309-CTLs have a potential role in promoting local inflammation within the CNS of HAM patients.

Effect of Accelerated Rehabilitation on Early Return to Sport After Arthroscopic Ankle Lateral Ligament Repair.

Background: Although the minimal invasiveness of arthroscopic ankle lateral ligament repair (ALLR) means that an early return to sporting activities can be anticipated, studies have described postoperative cast immobilization and the avoidance of weightbearing for a certain period. Accelerated rehabilitation may be helpful for an early return to sport. Purpose: To investigate clinical outcomes of ALLR and accelerated rehabilitation with a minimum duration of postoperative ankle immobilization and proactive early weightbearing. Study Design: Case series; Level of evidence, 4. Methods: This study investigated 23 ankles of 22 patients (11 men, 11 women; mean age, 38.7 years) who underwent ALLR for chronic lateral ankle instability. Postoperative management included the avoidance of weightbearing until postoperative day 3, after which full weightbearing walking with a brace was permitted. The objective was to return to competitive sport 8 weeks after surgery. The following were evaluated: pre- and postoperative instability and pain symptoms, ankle range of motion, anterior drawer distance on stress radiograph, anterior translation measured with a capacitance-type strain sensor, the Ankle-Hindfoot Scale from the Japanese Society for Surgery of the Foot, and the SAFE-Q (Self-Administered Foot Evaluation Questionnaire). Results: Two male patients dropped out and were excluded from analysis. Postoperatively, instability and pain resolved or improved in all patients. There was no significant postoperative change in range of motion. There were significant pre- to postoperative improvements in talar tilt angle (from 12.2°-5.6°, P < .01), anterior drawer distance (8.2-4.4 mm, P < .01), and anterior translation (10.5-4.6 mm, P < .01) as well as the Ankle-Hindfoot Scale score (68.8-96.8, P < .01) and all subscales of the SAFE-Q (P ≤ .01 for all). Complete return to sport was achieved by 75% of the patients at 8 weeks postoperatively. Conclusion: When accelerated rehabilitation with proactive weightbearing exercises was implemented from postoperative day 3 without ankle immobilization after ALLR, there were significant improvements in objective assessments of ankle stability and clinical scores, and as many as 75% of the patients were able to make a complete return to sport within 8 weeks.

Comparison of Treatment Methods for Syndesmotic Injuries With Posterior Tibiofibular Ligament Ruptures: A Cadaveric Biomechanical Study.

Background: Studies on ankle syndesmosis have focused on anterior inferior tibiofibular ligament (AITFL) and interosseous membrane injuries; however, the characteristics of posterior inferior tibiofibular ligament (PITFL) ruptures remain unclear. Purpose/Hypothesis: This study evaluated the biomechanical characteristics of syndesmotic instability caused by PITFL injury and compared various treatment methods. We hypothesized that PITFL injury would lead to syndesmotic internal rotational instability and that the stability would be restored with suture tape (ST) PITFL augmentation. Study Design: Controlled laboratory study. Methods: Ten uninjured fresh-frozen cadaveric leg specimens were tested via forces applied to the external and internal rotation of the ankle joint. The fibular rotational angle (FRA) related to the tibia, anterior tibiofibular diastasis (aTFD), and posterior tibiofibular diastasis (pTFD) were measured using a magnetic tracking system. Six models were created: (1) intact, (2) AITFL injury; (3) AITFL + PITFL injury; (4) suture button (SB) fixation; (5) SB + anterior ST (aST) fixation; and (6) SB + aST + posterior ST fixation. The FRA, aTFD, and pTFD were statistically compared between the intact ankle and each injury or fixation model. Results: In the intact state, the changes in FRA and aTFD were 1.09° and 0.33 mm when external rotation force was applied and were 0.57° and 0.41 mm when internal rotation force was applied. In the AITFL injury model, the changes in FRA and aTFD were 2.38° and 1.51 mm when external rotation force was applied, which were significantly greater versus intact (P = .032 and .008, respectively). In the AITFL + PITFL injury model, the changes in FRA and pTFD were 2.12° and 1.02 mm when internal rotation force was applied, which were significantly greater versus intact (P = .007 and .003, respectively). In the SB fixation model, the change in FRA was 2.98° when external rotation force was applied, which was significantly higher compared with intact (P < .001). There were no significant differences between the SB + aST fixation model and the intact state on any measurement. Conclusion: PITFL injury significantly increased syndesmotic instability when internal rotation force was applied. SB + aST fixation was effective in restoring syndesmotic stability. Clinical Relevance: These results suggest that SB + aST fixation is sufficient for treating severe syndesmotic injury with PITFL rupture.

Effect of early mobilization in patients with stroke and severe disturbance of consciousness: Retrospective study.

OBJECTIVES: This study aimed to investigate the effectiveness and safety of early mobilization with a physiatrist and registered therapist Operating rehabilitation (PROr) for patients with stroke and severe disturbance of consciousness (DoC). MATERIALS AND METHODS: We retrospectively screened records from patients with stroke admitted to our hospital from January 2015 to June 2021. Eligible patients with severe DoC were classified into two groups: patients who received standard rehabilitation (control group) and patients who received PROr (PROr group). We studied longitudinal change in the level of consciousness using the Japan Coma Scale (JCS) during hospital stay and compared in-hospital mortality, the incidence of respiratory complication, and modified Rankin Scale of discharge between the two groups. RESULTS: Among the 2191 patients screened for inclusion, 16 patients were included in the PROr group, and 12 patients were included in the control group. Early mobilization was more promoted in the PROr group compared to the control group, but there were no significant differences in in-hospital mortality, the incidence of respiratory complication, or modified Rankin Scale at discharge between the two groups. In patients who survived during their hospital stay, JCS scores 2 weeks after the onset of stroke and JCS scores at discharge significantly improved from the start of rehabilitation in the PROr group, but not in the control group. CONCLUSIONS: Early mobilization provided with the PROr program appears to be a safe treatment and may contribute to the improvement of consciousness level for patients with acute stroke and severe DoC.

Efficacy of Corticosteroid Therapy for HTLV-1-Associated Myelopathy: A Randomized Controlled Trial (HAMLET-P).

Corticosteroids are most commonly used to treat HTLV-1-associated myelopathy (HAM); however, their clinical efficacy has not been tested in randomized clinical trials. This randomized controlled trial included 8 and 30 HAM patients with rapidly and slowly progressing walking disabilities, respectively. Rapid progressors were assigned (1:1) to receive or not receive a 3-day course of intravenous methylprednisolone in addition to oral prednisolone therapy. Meanwhile, slow progressors were assigned (1:1) to receive oral prednisolone or placebo. The primary outcomes were a composite of ≥1-grade improvement in the Osame Motor Disability Score or ≥30% improvement in the 10 m walking time (10 mWT) at week 2 for rapid progressors and changes from baseline in 10 mWT at week 24 for slow progressors. In the rapid progressor trial, all four patients with but only one of four without intravenous methylprednisolone achieved the primary outcome (p = 0.14). In the slow progressor trial, the median changes in 10 mWT were -13.8% (95% CI: -20.1--7.1; p < 0.001) and -6.0% (95% CI: -12.8-1.3; p = 0.10) with prednisolone and placebo, respectively (p for between-group difference = 0.12). Whereas statistical significance was not reached for the primary endpoints, the overall data indicated the benefit of corticosteroid therapy. (Registration number: UMIN000023798, UMIN000024085).

Surgically treated reactive arthritis of the ankle after COVID-19 infection: A case report.

A 37-year-old man developed right ankle pain and swelling six days after being diagnosed with coronavirus disease (COVID-19). Despite conservative treatment, his ankle symptoms persisted. Magnetic resonance imaging and computed tomography showed synovial hypertrophy and bone erosion in the ankle. Following arthroscopic synovectomy, performed 69 days after the COVID-19 diagnosis, the pain improved significantly. The clinical course was consistent with that of reactive arthritis following severe acute respiratory syndrome coronavirus 2 infection. The pathological findings resembled rheumatoid nodules. The bone erosion may have originated from the inflammatory pathway, which resembles the mechanism of rheumatoid arthritis.

Relationship Between Plantar Callosity and Foot Deformity in Hallux Valgus Using Weightbearing Computed Tomography.

Plantar callosities under lesser metatarsals are often accompanied by the hallux valgus, and the cause of callosity is thought to be associated with the foot deformity, such as the metatarsal length discrepancy, the abnormal metatarsal head height, cavus, flat foot, and rheumatoid conditions. However, it is unclear which variable is most involved in the cause of callosity in hallux valgus deformity. To clarify the factors associated with the callosity with hallux valgus deformity, we conducted multiple image assessments based on weightbearing radiography and computed tomography. A retrospective review was performed based on the collection of clinical records from all patients with hallux valgus treated from 2010 to 2019 in our institution. We measured the hallux valgus angle, intermetatarsal angles, calcaneal pitch angles, talo-first metatarsal angles, metatarsal length, metatarsal head height, first metatarsal pronation angles, and sesamoid position with weightbearing radiography and computed tomography. We analyzed the relation between callosity formation and imaging assessments using univariate and multivariate logistic regression models. Fifty feet were retrospectively evaluated, and multiple logistic analyses by the stepwise method revealed that the first metatarsal-lateral-sesamoid distance was the only radiographical variable associated with callosity formation among all the tested variables (p < .001). As the grade of the callosity became more severe, the lateral shift of the lateral sesamoid increased. The position of the sesamoid bone appears to have a critical role in the assessment and choice of treatment protocols and further research needs to be conducted on the relationship with the position of sesamoid bone to elucidate the mechanism of callus formation.

Two New Terpenes Isolated from Dictyostelium Cellular Slime Molds.

We report a protoilludane-type sesquiterpene, mucoroidiol, and a geranylated bicyclogermacranol, firmibasiol, isolated from Dictyostelium cellular slime molds. The methanol extracts of the fruiting bodies of cellular slime molds were separated by chromatographic methods to give these compounds. Their structures have been established by several spectral means. Mucoroidiol and firmibasiol are the first examples of more modified and oxidized terpenoids isolated from cellular slime molds. Mucoroidiol showed moderate osteoclast-differentiation inhibitory activity despite demonstrating very weak cell-proliferation inhibitory activity. Therefore, cellular slime molds produce considerably diverse secondary metabolites, and they are promising sources of new natural product chemistry.

Halogen-Substituted Derivatives of Dictyostelium Differentiation-Inducing Factor-1 Suppress Serum-Induced Cell Migration of Human Breast Cancer MDA-MB-231 Cells in Vitro.

Triple-negative breast cancer (TNBC) is highly proliferative and metastatic, and because it lacks three major molecular targets for chemotherapy (estrogen receptor, progesterone receptor, and human epidermal receptor 2), it is extremely refractory. Differentiation-inducing factor 1 (DIF-1) and DIF-3, which are chlorinated alkylphenones, are lead anticancer compounds found in the cellular slime mold Dictyostelium discoideum. Here, we examined the in vitro effects of DIF-1, DIF-3, and 25 DIF derivatives on cell proliferation and serum-induced cell migration in human MDA-MB-231 cells, a model TNBC cell line. We found that Br-DIF-1, a chlorine-to-bromine-substituted derivative of DIF-1, strongly suppressed cell migration (IC50, 3.8 M) with negligible effects on cell proliferation (IC50, >20 M). We then synthesized 18 derivatives of Br-DIF-1 and examined the in vitro effects of these derivatives on cell proliferation and serum-induced cell migration in MDA-MB-231 cells. Among the derivatives, Br-DIF-1(+1), Br-DIF-1(+2), and Br-DIF-3(+2) exhibited strong anti-cell migration activities with IC50 values of 1.5, 1.0, and 3.1 M, respectively, without affecting cell proliferation (IC50, >20 M). These results suggest that these Br-DIF derivatives are good lead compounds for the development of anti-metastatic drugs against TNBC.

Antimicrobial Activities of Dictyostelium Differentiation-Inducing Factors and Their Derivatives.

At the end of its life cycle, the cellular slime mold Dictyostelium discoideum forms a fruiting body consisting of spores and a multicellular stalk. Originally, the chlorinated alkylphenone differentiation-inducing factors (DIFs) -1 and -3 were isolated as stalk cell inducers in D. discoideum. Later, DIFs and their derivatives were shown to possess several biologic activities including antitumor and anti-Trypanosoma properties. In this study, we examined the antibacterial activities of approximately 30 DIF derivatives by using several bacterial species. Several of the DIF derivatives strongly suppressed the growth of the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis and Enterococcus faecium, at minimum inhibitory concentrations (MICs) in the sub-micromolar to low-micromolar range. In contrast, none of the DIF derivatives evaluated had any noteworthy effect on the growth of the Gram-negative bacterium Escherichia coli (MIC, >100 µM). Most importantly, several of the DIF derivatives strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant E. faecalis and E. faecium. Transmission electron microscopy revealed that treatment with DIF derivatives led to the formation of distinct multilayered structures consisting of cell wall or plasma membrane in S. aureus. The present results suggest that DIF derivatives are good lead compounds for developing novel antimicrobials.

Aggregation Behavior and Thiol-Thioester Exchange for Cationic Surfactants with Propylthioacetate Side Chain.

A series of cationic surfactants containing the thioacetate group, [CnH2n+1N(CH3)2(CH2)3SCOCH3] Cl (Cn3SAc, n = 12, 14, 16), were prepared and their properties in aqueous solution were investigated by conductivity, fluorescence, and dynamic light scattering measurements. The critical micelle concentrations (CMCs) of Cn3SAc decreased to about half the value of the corresponding alkyltrimethylammonium chloride. Thioacetate was eliminated with the addition of dithiothreitol (DTT) as well as NaOH. HPLC (high performance liquid chromatography) analysis and NMR (nuclear magnetic resonance) spectroscopy showed that thiol surfactants, [CnH2n+1N(CH3)2(CH2)3SH]Cl (Cn3SH), were generated upon the addition of DTT in aqueous solution via thiol-thioester exchange, whereas gemini surfactants, [CnH2n+1N(CH3)2(CH2)3SS(CH2)3N(CH3)2CnH2n+1]2Cl (2Cn3SS), were generated upon incubation in alkaline solution via hydrolysis and air oxidation.

Biological Activities of Novel Derivatives of Differentiation-Inducing Factor 3 from Dictyostelium discoideum.

Differentiation-inducing factor-3 (DIF-3; 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), which is found in the cellular slime mold Dictyostelium discoideum, is a potential candidate compound for the development of new medicines; DIF-3 and its derivatives possess several beneficial biological activities, including anti-tumor, anti-Trypanosoma cruzi, and immunoregulatory effects. To assess the relationship between the biological activities of DIF-3 and its chemical structure, particularly in regard to its alkoxy group and the length of the alkyl chains at the acyl group, we synthesized two derivatives of DIF-3, 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)octan-1-one (DIF-3(+3)) and 1-(3-chloro-2,6-dihydroxy-4-butoxyphenyl)-hexan-1-one (Hex-DIF-3), and investigated their biological activities in vitro. At micro-molar levels, DIF-3(+3) and Hex-DIF-3 exhibited strong anti-proliferative effects in tumor cell cultures, but their anti-T. cruzi activities at 1 µM in vitro were not as strong as those of other known DIF derivatives. In addition, Hex-DIF-3 at 5 µM significantly suppressed mitogen-induced interleukin-2 production in vitro in Jurkat T cells. These results suggest that DIF-3(+3) and Hex-DIF-3 are promising leads for the development of anti-cancer and immunosuppressive agents.