Analyzing the evolution of insect TMED gene and the expression pattern of silkworm TMED gene / 生物工程学报

Transmembrane emp24 domain (TMED) gene is closely related to immune response, signal transduction, growth and disease development in mammals. However, only the Drosophila TMED gene has been reported on insects. We identified the TMED family genes of silkworm, Tribolium castaneum, tobacco moth and Italian bee from their genomes, and found that the TMED family gene composition patterns of one α-class, one β-class, one δ-class and several γ-classes arose in the common ancestor of pre-divergent Hymenoptera insects, while the composition of Drosophila TMED family members has evolved in a unique pattern. Insect TMED family γ-class genes have evolved rapidly, diverging into three separate subclasses, TMED6-like, TMED5-like and TMED3-like. The TMED5-like gene was lost in Hymenoptera, duplicated in the ancestors of Lepidoptera and duplicated in Drosophila. Insect TMED protein not only has typical structural characteristics of TMED, but also has obvious signal peptide. There are seven TMED genes in silkworm, distributed in six chromosomes. One of seven is single exon and others are multi-exons. The complete open reading frame (ORF) sequences of seven TMED genes of silkworm were cloned from larval tissues and registered in GenBank database. BmTMED1, BmTMED2 and BmTMED6 were expressed in all stages and tissues of the silkworm, and all genes were expressed in the 4th and 5th instar and silk gland of the silkworm. The present study revealed the composition pattern of TMED family members, their γ class differentiation and their evolutionary history, providing a basis for further studies on TMED genes in silkworm and other insects.

A novel PI3K inhibitor XH30 suppresses orthotopic glioblastoma and brain metastasis in mice models

Glioblastoma is carcinogenesis of glial cells in central nervous system and has the highest incidence among primary brain tumors. Brain metastasis, such as breast cancer and lung cancer, also leads to high mortality. The available medicines are limited due to blood-brain barrier. Abnormal activation of phosphatidylinositol 3-kinases (PI3K) signaling pathway is prevalent in glioblastoma and metastatic tumors. Here, we characterized a 2-amino-4-methylquinazoline derivative XH30 as a potent PI3K inhibitor with excellent anti-tumor activity against human glioblastoma. XH30 significantly repressed the proliferation of various brain cancer cells and decreased the phosphorylation of key proteins of PI3K signaling pathway, induced cell cycle arrest in G1 phase as well. Additionally, XH30 inhibited the migration of glioma cells and blocked the activation of PI3K pathway by interleukin-17A (IL-17A), which increased the migration of U87MG. Oral administration of XH30 significantly suppressed the tumor growth in both subcutaneous and orthotopic tumor models. XH30 also repressed tumor growth in brain metastasis models of lung cancers. Moreover, XH30 reduced IL-17A and its receptor IL-17RA in vivo. These results indicate that XH30 might be a potential therapeutic drug candidate for glioblastoma migration and brain metastasis.

Metabolomic alterations of human lung bronchial epithelial cells infected with human rhinovirus 1B / 中华微生物学和免疫学杂志

Objective:To research the metabolomic alterations of human lung bronchial epithelial cells infected with human rhinovirus 1B (HRV1B).Methods:Untargeted metabolomics was used to determine the metabolomic alterations in human lung bronchial epithelial cells (BEAS-2B) 6 h, 12 h and during the dynamic process (6 h∶12 h) after HRV1B infection.Results:A total of 93 differentially significant metabolites (DSMs) (47 DSMs were up-regulated and 46 DSMs were down-regulated) and 88 DSMs (37 DSMs were up-regulated and 51 DSMs were down-regulated) at post infection of HRV1B in BEAS-2B at 6 h or 12 h, respectively. A total of 30 DSMs (12 DSMs were up-regulated and 18 DSMs were down-regulated) in a dynamic process (6 h∶12 h) after HRV1B infection. Unknown metabolites took up most proportions. The trends of fatty acid, lipid, amino acid, nucleotide and carbohydrate were increased along with the prolonging of HRV1B infection. DSMs such as Diisononyl phthalate was co-detected DSMs among three groups.Conclusions:Metabolites such as fatty acid, lipid, amino acid, nucleotide and carbohydrate of BEAS-2B cells are changed induced by HRV1B infection.

Clinical analysis of 10 patients of pregnancy with cervical cancer / 中国医师进修杂志

Objective:To analyze the clinical features, diagnosis and treatment plan, clinical outcomes of pregnancy with cervical cancer.Methods:The clinical data of 10 pregnant women with cervical cancer from January 2008 to October 2018 in Dalian Obstetrics and Gynecology Hospital Affiliated to Dalian Medical University, the First Hospital of Dalian Medical University, Dalian Central Hospital, Dalian Women and Children′s Medical Center and Wafangdian Central Hospital of Liaoning Province were retrospectively analyzed.Results:The incidence of pregnancy with cervical cancer was 0.004% (10/238 128). Among the 10 cases of pregnancy with cervical cancer, the gestational weeks ≤ 20 weeks at the time of diagnosis was in 6 cases, and they all chose to terminate the pregnancy; the gestational weeks 20 +1 to 30 weeks at the time of diagnosis was in 1 case, and the patient chose to terminate the pregnancy; the gestational weeks >30 weeks at the time of diagnosis was in 2 cases, and they all chose to continue the pregnancy; 1 case was diagnosed after delivery. There were 3 newborns, including 1 premature infants, and they all survived. Conclusions:It is helpful to the diagnose of the disease to strengthen cervical cancer screening before pregnancy and improve the examination of patients with abnormal symptoms during pregnancy. The treatment plan should be individualized according to the pregnancy status, stage of the disease, and wishes of the patient and family.

Screening and confirmation of TP antibody and analysis of syphilis infection status among 12 295 blood donors in Huainan area / 中国基层医药

Objective:To understand the current situation of syphilis infection among 12 295 blood donors in Huainan area, so as to provide reference for the management of public health.Methods:The Treponema pallidum(TP) antibody of the blood donors was screened by enzyme-linked immunosorbent assay, and the unqualified blood donors for TP antibody were confirmed by Treponema pallidum particle agglutination(TPPA).Results:There were 48 blood donors TPPA positive among 12 295 blood donors in Huainan area, and the TPPA positive rate was 3.90‰(48/12 295). There was no statistically significant difference in the TPPA positive rate of the different gender blood donors[male 3.12‰(22/7 048) vs.female 4.96‰(26/5 247)] in Huainan area( u=1.61, P>0.05). There was statistically significant difference in the TPPA positive rate of the different marriage blood donors[married 5.33‰(42/7 884) vs.unmarried 2.01‰(6/2 989)] in Huainan area( u=2.33, P<0.05). There was statistically significant difference in the TPPA positive rate of the blood donors among different education level in Huainan area(χ 2=29.94, P<0.05). There was no statistically significant difference in the TPPA positive rate of the blood donors among ABO blood groups in Huainan area(χ 2=2.24, P>0.05). Conclusion:There are significant differences in the TPPA positive rates of the blood donors among marital status and education level in Huainan area.

Clinical application of posterior tibial artery perforator flap within saphenous nerve branch for sensory reconstruction / 中华显微外科杂志

Objective To explore the operative technique and clinical results of posterior tibial artery perforator flap within saphenous nerve branch for sensory reconstruction.Methods From January,2016 to June,2018,9 patients suffered from soft tissue defect were treated by the posterior tibial artery perforator flap containing saphenous nerve branch.Seven patients were males and 2 were females,with age ranged from 31 to 62 years.Soft tissue defects located in hands in 5 patients,plantar in 2 patients,ankle in 1 patient and dorsal foot in 1 patient.The size of soft tissue defects ranged from 8.0 cm×2.5 cm to 21.0 cm×4.0 cm.The regular post-operative followed-up was performed.Results All flaps survived without complications.The size of flap ranged from 10.0 cm×3.5 cm-23.0 cm×5.0 cm.Donor sites were primarily closed in 5 patients and secondary closed in 4 patients.Followed-up ranged from 6 to 15 months with 10 months in average.The contour of flaps were satisfied and the sensory function of the donor sites were normal.At 6 months followed-up,SW test reached 5.07 in all flaps,and 2PD ranged from 14 to 35 mm.Conclusion The novel sensory flap can provide satisfied sensory outcome without sacrificing main artery and saphenous nerve,and is a good candidate for sensory reconstruction of soft tissue defects.

Internal fixation combined with bone grafting for distal humeral nonunion / 中华创伤骨科杂志

Objective To evaluate the outcomes of internal fixation combined with bone grafting in the treatment of distal humeral nonunion.Methods This retrospective study included 48 patients who had undergone internal fixation combined with iliac bone grafting for distal humeral nonunion between January 2011 and December 2015 at Department of Orthopedic Surgery,The Sixth People's Hospital of Shanghai.They were 31 males and 17 females,with a mean age of 35.4 years (from 22 to 49 years).The outcomes were evaluated by clinical examination,X-ray film,Mayo elbow performance score (MEPS) and visual analogue scale (VAS) during follow-up.Results All the 48 patients were followed up for 18 to 60 months (mean,36 months).No wound-related complications were reported in this cohort.Solid bone union was achieved at 3 to 8 months (mean,4.2 months) after surgery.At the last follow-up,the range of elbow flexion-extension was improved from preoperative 66° to 101°,and the ranges of pronation and supination were enhanced from 65° and 45° preoperatively to 82° and 75°,respectively.The MEPS increased from 54 points preoperatively to 82 points postoperatively.The results were excellent in 26,good in 16 and fair in 6 cases (with an excellent to good rate of 87.5％).The VAS decreased from 4.5 points preoperatively to 1.2 points postoperatively,indicating notable relief of the elbow pain due to distal humeral nonunion.Follow-ups revealed no ulnar nerve injury,nonunion,heterotopic ossification,implant loosening or breakage,or instability of the elbow joint.Conclusion Internal fixation combined with bone grafting is reliable in treatment of distal humeral nonunion,leading to satisfactory outcomes.

Free chimeric vascularized fibular graft and sural flap for reconstruction of composite extremity defects / 中华创伤骨科杂志

Objective To report clinical application of free chimeric vascularized fibular graft combined with sural flap for reconstruction of composite extremity defects after open fracture.Methods From June 2010 to July 2014,free chimeric vascularized fibular grafts and sural flaps were used to treat 4 patients with composite extremity defects at Department of Orthopaedics,The Sixth People's Hospital.They were 3 men and one woman,aged from 39 to 61 years(average,48.5 years).There were 2 cases of soft tissue defects on the forearm complicated with radial defect,one case of soft tissue defects on the forearm complicated with ulnar shaft defect,and one case of soft tissue defects on the leg complicated with tibial defect.The length of bone defect ranged from 8 cm to 18 cm (average,13.0 cm);the size of soft tissue defects ranged from 22 cm × 6cmto23cm × 15 cm (average,22.3cm × 9.7cm).Results The area of flap ranged from 25 cm × 9 cm to 26 cm × 18 cm (average,25 cm × 13 cm);the length of fibular graft ranged from 8 cm to 18 cm (average,13 cm).The 4 patients were followed up for 8 to 42 months (average,20.5 months).All the chimeric flaps survived.All the fractures united after an average of 8.5 months.The last follow-ups revealed no refracture.All the patients were satisfied with the outcomes.Conclusion Free chimeric vascularized fibular graft combined with sural flap is a reliable choice for reconstruction of composite extremity defects after open fracture.

Effect of different stimulants on the LAG3 expression and function of spleen lymphocytes in mice / 中国生化药物杂志

Objective To investigate the effect of different stimulants on the LAG3 expression and function of spleen lymphocytes in mice. Methods The spleen lymphocytes from mice were isolated by density centrifugation.The LAG3 expressions in T cell subsets after exposure to conA, PMA, PHA or anti-CD3/28 antibodies for 24 h or 72 h were analyzed by Flow cytometry.The IFN-γsecretions of conditional medium were detected by ELISA kit.The proliferation of lymphocytes was examined by MTT analysis.Results Treatment with conA for 24 h or 72 h dose-dependently increased LAG3 +CD3 +and LAG3 +CD4 +CD3 +T cell percentages.Similarly, an exposure of anti-CD3/28 antibodies for 72 h significantly increased LAG3 +CD3 +and LAG3 +CD4 +CD3 + T cell percentages.Meanwhile, conA and anti-CD3/28 antibodies increased the IFN-γsecretion of lymphocytes in a time-dependent manner.Furthermore, Treatment with conA, PMA, PHA or anti-CD3/28 antibodies for 72 h could enhance the proliferation of lymphocyte. Conclusion conA and anti-CD3/28 antibodies are effective activators of T cells, and both of them could promote the expression of LAG3 and IFN-γsecretion of lymphocytes.

Establishment of malignant pleural effusion model of Lewis lung cancer based on Micro Echo Technology / 中国生化药物杂志

Objective To establish a malignant pleural effusion model of Lewis lung cancer in C57BL/6 mice based on Micro Echo technology. Methods Single tumor cell suspension of Lewis lung cancer was injected into thoracic cavity.The pleural effusion was detected by Micro Echo technology.The volumes of effusion were quantified and the tumor cells were counted.Results After implanted of tumor cell, malignant pleural effusion can be detected by Micro Echo technology and observed after autopsy.Chemotherapy drugs such as Cyclophosphamide and Cisplatin can decrease the effusion volumes.Conclusion Pleural effusion model of Lewis lung cancer based on Micro Echo technology can be used to evaluate the efficacy of antitumor drugs.

Development of the high-throughput screening fluorescence polarization assay for HSP90 inhibitor / 中国生化药物杂志

Objective To establish the high-throughput screening fluorescence polarization assay for HSP90 inhibitor.Methods E.coli strain BL21 ( DE3) competent cells were transformed with pET24α( +)-HSP90αplasmid.The cell lysate supernatant was induced to product the soluble protein and purified with Ni-NTA agarose.Western blot analysis was used to identify whether the purified protein is HSP90α.The fluorescence polarization assay for screening HSP90 inhibitors was established and optimized using varying concentrations of recombinant HSP90 protein and molecular probe VER00051001.Meanwhile, the binding activity of GA and NVP-AUY922 for HSP90αwas measured by fluorescence polarization assay.Results HSP90αwas induced expression and purified successfully.The fluorescence polarization assay was performed using 80 nM probe VER00051001 and 2.01μg/mL HSP90α, with the Z factor of 0.83.GA and NVP-AUY922 competed with the probes VER00051001 for binding sites of HSP90, with IC50 of 55 nM and 13 nM, respectively.Conclusion A reliable model was established using fluorescence polarization assay for screening HSP90 inhibitors.

Inhibiting miR-29 on growth, invasion and metastasis of PANC1 cells / 中华胰腺病杂志

Objective To investigate the effects of inhibiting miR-29 on growth,invasion and metastasis of pancreatic cancer PANC1 cells,and explore the potential mechanism.Methods Oligonucleotides inhibiting miR-29 (anti miR-29) and control oligonucleotides (miR NC) were used to transfect PANC1 cells to establish anti miR-29 PANC1 cells and miR NC PANC1 cells.Transient transfection of PUMA siRNA,E-cadherin siRNA or NC siRNA was used to construct cotransfected anti miR29 + PUMA-siRNA-PANC1 cells and anti-miR-29 + E-cadherin-siRNA-PANC1 cells.Number of colony formations was observed,cell survival was detected by MTT,cell apoptosis was measured by flow cytometry,cell invasion was detected by transwell chamber assay,and cell migration was detected by wound healing assay.Subcutaneous injection of anti miR-29 PANC1 cells was used to establish xenograft nude mice model,and venous injection of anti miR-29 PANC1 cells was used to establish lung metastasis nude mice model,and the subcutaneous and venous injection of PANC1 cells served as control.The growth of xenograft and the number of lung metastatic nodules were observed.TUNEL method was used to detect cell apoptosis in xenograft and immunohistochemical analysis was used to detect PUMA and E-cadherin in xenograft.Results The survival rate of PANC1,miR-NC-PANC1 and anti-miR-29-PANC1 cells was 100％,(96.8 ± 2.8) ％ and (24.4 ± 3.2) ％.The number of colony formation was (213 ± 36),(196 ± 28) and (37 ± 6) per 100 high power field.The number of transmembrane cells was (56.3 ± 9.6),(49.8-± 7.3) and (11.2 ± 3.4) per 400 high power field.The distance of cell migration was (260 ± 48),(247 ± 46) and (53 ± 7) μm.Cell apoptosis rate was (1.5 +0.9) ％,(2.6 + 0.9) ％ and (22.4 + 2.8) ％.There was statistically significant difference between anti miR 29 PANC1 cells and other PANC1 cells (P ＜0.05).The survival rate,apoptosis rate,transmembrane cells and migration distance of anti-miR-29 + PUMA-siRNA-PANC1 cells was (84.7 ± 10.9) ％,(1.3 ± 0.8) ％,(49.7 ± 6.4) per 400 high power field and (182 ± 36) μm,indicating that the effects of miR 29 inhibition on PANC1 cells were abolished (all P ＜0.05).The volume of the xenograft of PANC1 and anti-miR-29-PANC1 cells was (3 800 ±270) and (1 890 ± 160)mm3,the cell apoptosis rate was 0.93 ±0.14 and 8.26 ± 1.15,the number of metastatic lung lesions was (26.4 ± 6.5) and (8.6 ± 2.7),the PUMA positivity was (7.2 ±1.6) ％ and (43.8 ± 7.6) ％,E-cadherin positivity was (8.3 ± 3.6) ％ and (47.4 ± 5.7) ％,respectively.The xenograft volume and the number of metastatic lung nodules of anti miR29 PANC1 cells was obviously decreased or decreased,but cell apoptosis rate,PUMA positivity and E cadherin positivity were obviously increased,and the differences were all statistically significant (P ＜ 0.05).Conclusions Inhibiting miR-29 expression can decrease cell proliferation,migration and metastasis of PANC1 cells,and the potential mechanism may be associated with the upregulation of PUMA and E-cadherin.

Application progress of disease modifying drugs in neuromyelitis optica spectrum disorder / 天津医药

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease of the central nervous system that is characterized by inflammatory demyelinating lesions and axonal degeneration in optic nerve and spinal cord. It has high relapse rate and high disability rate. In recent years, as the studies on mechanisms of NMOSD, related immune therapy strategies have been developed rapidly. These immunologic interventions are collectively called the disease modifying therapy, which are helpful in improving the course and outcome of the disease. In this review, the latest treatment progress in NMOSD is summarized to guide clinical choice and application of the medication.

Clinical application of the reversed saphenous fasciocutaneous island flap combined with the vacuum sealing drainage therapy in plate exposure of the distal tibia / 中华显微外科杂志

Objective To evaluate the clinical effect of the reversed saphenous fasciocutaneous island flap combined with the VSD therapy in plate exposure of the distal tibia.Methods From January 2008 to July 2010,seven patients with hardware exposure following internal fixation of the distal tibia fracture were treated by VSD therapy and reversed saphenous fasciocutaneous island flap reconstruction.The VSD therapy was performed from 7 to 26 days after hardware exposure,and the flap transfer was performed 7 to 14 days after the VSD therapy.The size of the soft-tissue defect ranged from 4 cm × 2 cm to 13 cm × 4 cm.The mean follow-up was 10.6 months after the fracture (range,8-14 months).Results The average times of the VSD therapy was 1.3.After VSD was removed,the exposed hardware was covered by healthy granulation tissue in all cases.All flaps were successfully transplanted,and the size of flap ranged from 6 cm × 3 cm to 15 cm × 6 cm.Six of 7 flaps survived completely without further procedures.The necrosis of the distal margin of the flap occurred in 1 patient.A solid bony union was confirmed by the X-ray in all patients after 4-6 months postoperatively.No skin defects or fistulae were observed.Conclusion The combination of the reversed saphenous fasciocutaneous island flap and VSD therapy could save the exposed hardware and cover the soft tissue defects in the distal tibia.

The free peroneal perforator-based sural neurofasciocutaneous flap: anatomical study and clinical application / 中华显微外科杂志

Objective To report the anatomical study and clinical application of the free peroneal perforator-based sural neurofasciocutaneous flap. Methods Latex injection studies were performed on 10 adult cadaveric lower limbs. The presence, prevalence, and location of the peronel perforators that were suitable for vascular anastomosis were documented. From April 2007 to January 2010, 6 patients with large soft tissue defects in the upper limb underwent the reconstructive procedures with the free peroneal perforatorbased sural neurofasciocutaneous flap transfers. 4 patients were men and 2 were women, with the age ranging from 19 to 60 years. The causes of the injury included 4 motor vehicle accidents, and 2 massive machinery trauma. The area of the flap ranged from 16 cm × 8 cm to 30 cm × 10 cm, and the length of the perforator pedicle was 4 cm to 6 cm. Results A mean of 5.3 perforating vessels of the peroneal artery was noted in the specimen. The peroneal perforator that located at the junction of the middle and lower thirds of the fibula was found suitable for microsurgery, with the external diameter and the length being (1.21 ± 0.13)mm and (4.6 ± 0.8)cm respectively. All the 6 flaps survived completely without necrosis. No severe venous congestion was observed. All the patients were satisfied with the aesthetic outcome postoperatively at 3 to 12 months'follow-up. There were no major donor site complications. Conclusion The free sural neurofasciocutaneous flap based on a single peroneal perforator is an excellent tool for reconstruction of extensive soft tissue defects in the upper limb.

Anatomy and application of free medial plantar perforator flap / 中华显微外科杂志

Objective To reports to anatomy and clinical application of the medialis pedis perforator flap. Methods The origin, course, distribution and out diameter of medial plantar perforators, which were located at the septums between the abductor hallucis muscle and the flexor digitorum brevis, and between the abductor hallucis muscle and the skeleton, were observed on 10 sides adult feet specimens perfused with red latex. 11 free medialis pedis perforator flaps were transferred for soft-tissue defect in hand. The areas of tissue defect ranged from 2 cm x 2 cm - 9 cmx 4 cm. Results The medial plantar artery sends 2 perforators with regular anatomy through the septum between the abductor hallucis muscle and the flexor digitomm bre-vis, and 2 perforators with regular anatomy through the septum between the abductor hallucis muscle and nav-icluar bone and the medial cuneiform bone. These perforators supply the medial plantar flap and medialis pedis flap respectively. All of the 11 cases of free medialis pedis perforator flap survived uneventfully. The flap areas ranged from 2 cm × 3 cm - 11 cm× 5 cm. The appearance and functional results were satisfactory with following up for 6 to 24 months. Conclusion The free medialis pedis perforator flap is a good method in repairing soft-tissue defect in hand.

Anatomical study and clinical application of the vascularized composite fibula perforating osteoseptocutaneous flap / 中华显微外科杂志

@#Objective To report the anatomical study and clinical application of the vascularized composite fibula perforating osteoseptocutaneous flap. Methods Latex injection studies were performed on 24 adult cadaveric lower limbs. The presence, prevalence, and location of the peronel artery and its perforators in the distal leg were documented. 16 vascularized fibul osteoseptocutaneous flaps were performed for reconstruction of composite soft-tissue defect of limbs. The flap areas ranged from 6 cm×4 cm to 16 cm× 8 cm. Results A mean of 5.3 perforating vessels of the peroneal artery was noted in the specimen. 40.6% of them were muscularcutanous perforators, and the other were septacutanous perforators. The perforating vessels were all located within 3.1 cm from the postlateral fibula. The average diameter was 1.1 mm at the deep fascial level, and the interval of them was 4-8 cm. There were two constant perforators which located about 15-25 cm and 4-7 cm from the lateral maleolar respectively. All of the 16 clinical cases survived uneventfully and the time to union of the graft-host junction site were 3 to 5 months. The appearance of flaps and the function of the limbs were satisfied during 10 to 36 months following up. Conclusion It is the anatomic basis that there are pedorating vessels of the peroneal constantly. The vnscularized compound fibula perforating osteoseptocutaneous flap has the advantage of flexible design. It is the safe and suitable choice in the cases when compound bone and soft tissue defects of the limbs are required for reconstruction.

Metabolism of Escherichia coli is interfered by Bacillus subtilis glnA gene / 生物工程学报

A recombinant strain Escherichia coli DH5alpha(pMD19-glnA) including Bacillus subtilis glnA gene was constructed. Capillary electrophoresis and nuclear magnetic resonance were used to determine qualitatively the product of transformation by recombinant strain, and the relative level of mRNA expression of glnA was also determined by fluorescence quantitative RT-PCR. Subsequently, SDS-PAGE (polyacrylamide gel electrophoresis) was used to analysis the relative level of protein. Surprisingly, there was no increase of glutamine production in this recombinant strain, but an obvious increase in the GABA (gamma-aminobutyric acid ) production. It was showed in the experiment that protein expression of the glutamine synthetase did not increase, although glnA gene can be transcribed normally in this recombined strain. The phenomenon of exogenous glnA gene interfering metabolism of Escherichia coli was worthy of further study.