Efficient detection of Streptococcus pyogenes based on recombinase polymerase amplification and lateral flow strip.

PURPOSE: This article aims to establish a rapid visual method for the detection of Streptococcus pyogenes (GAS) based on recombinase polymerase amplification (RPA) and lateral flow strip (LFS). METHODS: Utilizing speB of GAS as a template, RPA primers were designed, and basic RPA reactions were performed. To reduce the formation of primer dimers, base mismatch was introduced into primers. The probe was designed according to the forward primer, and the RPA-LFS system was established. According to the color results of the reaction system, the optimum reaction temperature and time were determined. Thirteen common clinical standard strains and 14 clinical samples of GAS were used to detect the selectivity of this method. The detection limit of this method was detected by using tenfold gradient dilution of GAS genome as template. One hundred fifty-six clinical samples were collected and compared with qPCR method and culture method. Kappa index and clinical application evaluation of the RPA-LFS were carried out. RESULTS: The enhanced RPA-LFS method demonstrates the ability to complete the amplification process within 6 min at 33 °C. This method exhibits a high analytic sensitivity, with the lowest detection limit of 0.908 ng, and does not exhibit cross-reaction with other pathogenic bacteria. CONCLUSIONS: The utilization of RPA and LFS allows for efficient and rapid testing of GAS, thereby serving as a valuable method for point-of-care testing.

[Cervical spondylosis of nerve root type with qi stagnation and blood stasis treated with warming needle with different lengths of moxa stick: a randomized controlled trial].

OBJECTIVE: To compare the clinical efficacy on cervical spondylosis of nerve root type with qi stagnation and blood stasis treated with warming needle with different lengths of moxa stick. METHODS: Six hundred patients with cervical spondylosis of nerve root type with qi stagnation and blood stasis were randomly divided into 4 groups: a 4 cm length group (150 cases, 5 cases dropped off, 2 cases suspended), a 3 cm length group (150 cases, 6 cases dropped off, 2 cases suspended), a 2 cm length group (150 cases, 6 cases dropped off), and a routine acupuncture group (150 cases, 6 cases dropped off). Warming needle with moxa stick in the length of 4 cm, 3 cm and 2 cm was delivered in the 4 cm length group, the 3 cm length group and the 2 cm length group, respectively. In the routine acupuncture group, simple acupuncture was applied. The acupoints selected in the above groups included Dazhui (GV 14) and bilateral Jiaji (EX-B 2) of C5 and C7, Fengchi (GB 20), Jianzhen (SI 9), Quchi (LI 11), Zhongzhu (TE 3), etc. In each group, the intervention was delivered once daily and 5 times a week. One course of intervention was composed of 2 weeks and 2 courses were required. The TCM syndrome score, the score of clinical assessment scale for cervical spondylosis (CASCS), the score of the brachial plexus traction test of the affected upper limb, F wave occurrence rate and conduction velocity of the ulnar nerve, the median nerve and the radial nerve of the affected upper limb were compared before and after treatment in the patients of each group. The levels of serum inflammatory factors, i.e. interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and hypersensitive C-reactive protein (hs-CRP), were measured before and after treatment in the patients of each group. The clinical cfficacy was evaluated in the 4 groups. RESULTS: After treatment, the results of TCM syndrome evaluation, i.e. the scores of neck pain, activity limitation and upper limb numbness and pain, as well as the total scores; and the scores of brachial plexus traction test were reduced when compared with those before treatment in each group (P<0.01, P<0.05). The scores of subjective symptoms and adaptability, and the total scores of CASCS were elevated in comparison with those before treatment in each group (P<0.01, P<0.05). In the 4 cm length group, compared with the other 3 groups, the scores of neck pain and activity limitation for TCM syndrome evaluation, and its total score were lower (P<0.05, P<0.01); and the scores of subjective symptoms and adaptability, and the total score of CASCS were higher (P<0.05, P<0.01). The score of the brachial plexus traction test in the 4 cm length group was lower than that of the routine acupuncture group (P<0.05). After treatment, F wave occurrence rates and conduction velocity of median nerve and radial nerve were increased when compared with those before treatment in each group (P<0.05, P<0.01). F wave occurrence rate and conduction velocity of the radial nerve in the 4 cm length group were higher than those of the other 3 groups (P<0.05), and those of the median nerve were higher when compared with the routine acupuncture group (P<0.05). After treatment, the levels of serum IL-1ß, IL-6 and TNF-α were all reduced when compared with those before treatment in each group (P<0.01, P<0.05); the level of serum IL-6 in the 4 cm length group was lower than those of the other 3 groups and serum level of TNF-α was lower compared with that in the routine acupuncture group (P<0.05). The total effective rate of the 4 cm length group was 78.3% (112/143), which was higher when compared with the 3 cm length group (67.6%, 96/142), the 2 cm length group (65.3%, 94/144) and the routine acupuncture group (53.5%, 77/144), respectively (P<0.05). CONCLUSION: Warming needle with moxa stick of 4 cm in length effectively relieves the clinical symptoms of cervical spondylosis of nerve root type with qi stagnation and blood stasis, improves the nerve function of the upper limbs, and reduces the inflammatory responses caused by nerve compression. The clinical efficacy of this therapy with moxa stick of 4 cm in length is superior to the warming needle with moxa sticks of 3 cm and 2 cm, as well as the routine acupuncture.

Downregulation of miR-221-3p promotes the ferroptosis in gastric cancer cells via upregulation of ATF3 to mediate the transcription inhibition of GPX4 and HRD1.

BACKGROUND: Gastric cancer (GC) is an aggressive gastrointestinal tumor. MiRNAs participate in the tumorigenesis of GC. Nevertheless, the function of miR-221-3p in GC remains largely unknown. METHODS: RNA levels were assessed by RT-qPCR. Western blot was performed to test the protein levels. The relation between miR-221-3p and ATF3 was investigated by dual-luciferase reporter assay. ChIP and dual-luciferase reporter assay were applied to assess the interaction between ATF3 and HRD1 or GPX4. Meanwhile, cell proliferation was investigated by CCK8 and colony formation assay. The content of erastin-induced Fe2+ was investigated by iron assay kit. Erastin-induced lipid ROS level was assessed by C11-BODIPY 581/591. Co-immunoprecipitation was used to detect the interaction between HRD1 and ACSL4. In addition, xenograft mice model was established to detect the effect of miR-221-3p in GC. RESULTS: Depletion of miR-221-3p greatly attenuated GC cell proliferation through promoting ferroptosis. Meanwhile, ATF3 was downregulated in GC, and it was identified to be the downstream mRNA of miR-221-3p. MiR-221-3p downregulation could promoted the ferroptosis in GC cells through upregulation of ATF3. HRD1 mediates ubiquitination and degradation of ACSL4 to inhibit ferroptosis. ATF3 upregulation could reduce GC cell proliferation via downregulating the transcription of GPX4 and HRD1. Furthermore, downregulation of miR-221-3p markedly attenuated the growth of GC in mice. CONCLUSION: HRD1 mediates ubiquitination and degradation of ACSL4 to inhibit ferroptosis. MiR-221-3p depletion upregulates the ferroptosis in GC cells via upregulation of ATF3 to mediate the transcription inhibition of GPX4 and HRD1. Our study might provide a novel target for GC treatment.

[Warming-needle moxibustion with different lengths of moxa stick for asthenospermia with kidney deficiency and liver depression: a randomized controlled trial].

OBJECTIVE: To explore the clinical effect of warming-needle moxibustion with different lengths of moxa stick for asthenospermia with kidney deficiency and liver depression. METHODS: A total of 240 patients with asthenospermia of kidney deficiency and liver depression were randomly divided into a 4-cm group (moxibustion with 4-cm moxa stick, 60 cases, 3 cases dropped off), a 3-cm group (moxibustion with 3-cm moxa stick, 60 cases, 4 cases dropped off), a 2-cm group (moxibustion with 2-cm moxa stick, 60 cases, 2 cases dropped off) and an acupuncture group (60 cases, 3 cases dropped off). All patients were treated with warming-needle moxibustion with different lengths of moxa stick or conventicnal acupuncture at Zhongwan (CV 12), Guanyuan (CV 4), Zhongji (CV 3), Guilai (ST 29), Yaoyangguan (GV 3), Guanyuanshu (BL 26), etc., once a day, five times a week; 4-week treatment was taken as one course, a total of two courses of treatment were given. The semen routine indexes, seminal plasma biochemical indexes, sex hormone levels and TCM syndrome score were compared before and after treatment among the 4 groups. RESULTS: After treatment, the sperm density, sperm viability, ratio of grade A sperm, ratio of grade A and B sperm, seminal plasma fructose and neutral α-glucosidase were higher than those before treatment (P<0.05, P<0.01), and the sperm deformity rates were lower than those before treatment in the 4-cm group and the 3-cm group (P<0.01, P<0.05). The ratio of grade A sperm, ratio of grade A and B sperm, seminal plasma fructose and neutral α-glucosidase in the 4-cm group were higher than the other three groups (P<0.05, P<0.01), and the sperm deformity rate was lower than the other three groups (P<0.05). After treatment, except for dizziness and tinnitus score, each-domain score and total scores of TCM syndrome scale in the 4-cm group and the 3-cm group were lower than those before treatment (P<0.01, P<0.05). The each-domain score of depression, weak waist and knees, low sexual function and total score in the 4-cm group were lower than those in the other three groups (P<0.05, P<0.01). The total effective rate was 87.7% (50/57) in the 4-cm group, which was higher than 78.6% (44/56) in the 3-cm group, 77.6% (45/58) in the 2-cm group and 70.2% (40/57) in the acupuncture group (P<0.05, P<0.01). CONCLUSION: The warming-needle moxibustion with 4-cm moxa stick could effectively improve quality and motility of sperm and clinical symptoms in patients with asthenospermia of kidney deficiency and liver depression, which is superior to moxibustion with 3-cm, 2-cm moxa sticks and conventional acupuncture.