[Intraoperative neuromonitoring in surgery of cervical neurogenic tumors].

Objective: To investigate the application value of intraoperative motor nerve monitoring in cervical neurogenic tumor surgery. Methods: The efficacy of intraoperative neuromonitoring (IONM) was analyzed retrospectively in 18 patients, including 6 males and 12 females, aged from 15 to 74 years, treated in Affiliated Drum Tower Hospital, Medical School of Nanjing University from June 2019 to September 2022 who underwent total cystectomy of cervical neurogenic tumors under intraoperative nerve monitoring. Results: All 18 patients had complete tumor removal, including 8 patients with tumors from the vagus nerve and 10 patients with tumors from the brachial plexus nerve. Postoperative nerve functions were normal in patients with tumors from brachial plexus nerve, and incomplete vocal cord paralysis occurred in 2 patients with tumors from vagus vagus nerve. The total incidence of motor nerve injury was 11.1% (2/18). All patients were followed up for 6 to 45 months, with no tumor recurrence. Conclusion: Intraoperative neuromonitoring has significant values in surgery of cervical neurogenic tumors, which is helpful to remove completely the tumors on the basis of protecting the nerve functions to the maximum extent.

[Early to mid-term clinical outcomes of aortic valve repair in patients with bicuspid aortic insufficiency].

Objective: To examine the early to mid-term clinical outcomes of aortic valve repair in patients with bicuspid aortic insufficiency. Methods: This is a retrospective cohort study. The clinical data of 124 patients with BAV insufficiency who underwent aortic valve repair from January 2017 to June 2023 in the Department of Cardiovascular Surgery at Fuwai Hospital were analyzed retrospectively. There were 117 males and 7 females with an age of (38.1±12.7) years (range: 14 to 65 years). Depending on whether the aortic sinus was replaced or not, surgical approaches were divided into valve sparing root replacement (reimplantation, remodeling, modified remodeling) and isolated aortic valve repair (annuloplasty, isolated aortic valve leaflet repair). Perioperative and follow-up data were collected. Kaplan-Meier method was used to plot the curves of survival rate, free recurrence rate of massive aortic valve insufficiency and free re-operation rate, and Log-rank test was used for comparison between groups. Results: Among the surgeries, there were 47 cases of reimplantation, 8 cases of remodeling, 8 cases of modified remodeling, 48 cases of aortic annuloplasty (external annuloplasty in 22 cases, CV-0 annuloplasty in 26 cases), and 13 cases of isolated leaflet repair. Leaflet plication was the most used leaflet repair technique, used in 103 patients. The cardiopulmonary bypass time was (133.7±56.9) minutes (range: 48 to 461 minutes), and aortic cross-clamp time was (103.8±47.8) minutes (range: 25 to 306 minutes), with no surgical mortality. All patients underwent outpatient or telephone follow-up. The cumulative follow-up time was 340.3 person-years and the mean follow-up time was (M (IQR)) 34.0 (25.5) months (range: 3 to 76 months). The 5-year survival rate was 98.4%, the 5-year freedom from significant insufficiency rate was 93.4% and the 5-year freedom from aortic valve reoperation rate was 95.6%. The subgroup analysis revealed a significantly better freedom from the significant insufficiency rate in the aortic valve annular reduction group compared to the non-reduction group (P<0.01). Conclusions: Aortic valve repair in patients with bicuspid aortic insufficiency could obtain steady early to mid-term outcomes. Aortic annuloplasty can reduce the risk of recurrent aortic valve insufficiency in patients undergoing aortic repair.

[Exploring the factors affecting music-related quality of life in post-lingual deaf adults with cochlear implants].

Objective: To observe the feelings of listening to music and the importance of music in the daily life of post-lingual deaf adults with cochlear implants, and to explore the relevant influencing factors. Methods: This was a cross-sectional survey study. From January 2021 to August 2021,the Music-Related Quality of Life Scale was used to evaluate the music needs and music experiences of 63 post-lingual deaf adults who met the inclusion criteria, including 27 males and 36 females, aged (40.7±12.3) years, at the time of surgery (36.8±13.1) years, and with a preoperative hearing aid ineffective time of (3.9±5.8) years. Indicators analyzed included age, duration of ineffective preoperative hearing aid wear, preoperative music preference, duration of postoperative cochlear implant use, current hearing aid modality, and auditory rehabilitation outcomes. Whether the six factors mentioned above constituted an influence on the subjects' music listening was investigated using SPSS 25.0 statistical software. Results: All of the observations in the scale were correlated with a single factor. The two sub-dimensions of music experience section were related to the effect of auditory rehabilitation. In the importance section, the effect of auditory rehabilitation was the influential factor of the dimension of "participation importance", and the preoperative enjoyment of music was the relevant influential factor of the dimension of "perceived importance". There was a significant difference between the groups when they were grouped by the above factors (P value<0.05), while there was no statistically significance between the groups when they were grouped by other factors (P value>0.05). Conclusions: Post-lingual deaf adults show the need and attempt to listen to music after cochlear implantation. The effectiveness of auditory rehabilitation and the degree of music preference preoperatively are two important factors that influence music listening in implant recipients. Once the level of auditory communication has been restored to a certain degree, it is important to pay more attention to the needs of music for implant recipients and train them in time, especially for those with music preferences preoperatively.

[Early outcome of valve sparing aortic root replacement with partial upper sternotomy].

Objective: To examine the early outcome of valve sparing aortic root replacement with reimplantation technique (David procedure) with partial upper sternotomy. Methods: From April 2016 to April 2020, 31 patients underwent valve sparing aortic root replacement under partial upper sternotomy at Vascular Surgery Center, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. There were 28 males and 3 females, aging (44±13) years (range: 11 to 65 years). Preoperative aortic regurgitation was found greater than moderate in 15 patients, moderate in 6 patients and less than moderate in 10 patients. The diameter of aortic annulus was (26±3) mm (range: 21 to 34 mm), the diameter of aortic sinus was (51±6) mm (range: 41 to 68 mm), the diameter of ascending aorta was (43±8) mm (range: 26 to 62 mm). The preoperative ejection fraction was (65±4) % (range: 59% to 72%) and left ventricular end-diastolic diameter was (55±6) mm (range: 42 to 68 mm). All cases were treated with David Ⅰ procedure, including simple David procedure in 26 patients, David+ascending aorta and partial aortic arch replacement in 3 patients, David+thoracic endovascular aortic repair in 1 patient, David+stent elephant trunk implantation in 1 patient. Results: The operation time, cardiopulmonary bypass time and aortic cross-clamping time were (330±58) minutes (range: 214 to 481 minutes), (138±23) minutes (range: 106 to 192 minutes) and (108±17) minutes (range: 82 to 154 minutes), respectively. There were no death and serious complications (stroke, myocardial infarction, renal insufficiency, severe infection, etc.). The postoperative drainage volume within 24 hours was (314±145) ml (range: 130 to 830 ml). The intubation time was (14±3) hours (range: 8 to 21 hours), and the ICU time was (M(QR)) 2.1(1.5) days (range: 1.0 to 5.0 days). Eight patients had no blood transfusion, the proportion of red blood cell use was 9.7% (3/31), plasma use was 22.6% (7/31), and platelet use was 71.0% (22/31). The postoperative left ventricular ejection fraction was (62±4)% (range: 54% to 69%), and left ventricular end-diastolic diameter was (48±4) mm (range: 39 to 56 mm). After operation, aortic regurgitation was significantly improved, with no more than moderate regurgitation, small to moderate regurgitation in 3 patients, minor regurgitation in 3 patients, micro regurgitation in 12 patients and no regurgitation in 13 patients. The follow-up period was 3.5(6.1) months (range: 2.0 to 39.0 months). Echocardiographic follow-up data were obtained in 26 cases, including moderate regurgitation in 1 patient, small to moderate regurgitation in 9 patients, minor regurgitation in 5 patients, micro regurgitation in 6 patients and no regurgitation in 5 patients. There were no major adverse cardiovascular events and aortic events during the follow-up period. No patient was reoperated for aortic regurgitation. Conclusion: Valve sparing aortic root replacement under partial upper sternotomy is safe and feasible, and the early result is satisfactory.

[Discussion on the diagnosis and treatment of high-grade squamous intraepithelial lesions in post-menopausal women].

Objective: To analyze the performance of colposcopy and investigate the diagnosis and treatment characteristics of high-grade squamous intraepithelial lesion (HSIL) diagnosed by cervical tissue sampling in post-menopausal women. Methods: A retrospective study was performed on 1 449 patients with HSIL diagnosed by cervical tissue sampling under colposcopy and treated by loop electrosurgical excision procedure (LEEP) or extrafascial hysterectomy as the primary therapy at the First Affiliated Hospital of Nanjing Medical University, from November 2015 to October 2017. In order to investigate the diagnosis and treatment of HSIL in post-menopausal women, a case-control study was conducted to compare the difference in performance of colposcopy and treatment modality between 213 post-menopausal patients (14.7%, 213/1 449) and 1 236 pre-menopausal patients (85.3%, 1 236/1 449). Results: (1)The proportion of cases pathologically upgraded to cervical cancer was significantly greater in post-menopausal patients (9.4%, 20/213) compared with pre-menopausal patients (3.8%, 47/1 236; P<0.05). (2) The proportion of ≥HSIL diagnosed by colposcopy showed no significant difference between post-menopausal patients (76.1%, 162/213) and pre-menopausal patients (78.2%, 967/1 236; P=0.479). The proportion of type Ⅲ transformation zone (TZ) was significantly greater in post-menopausal patients (91.1%, 194/213) compared with pre-menopausal patients (59.1%, 731/1 236; P<0.05). The rate of missed diagnosis of cervical cancer was significantly higher in type Ⅲ TZ (6.4%, 59/925) compared with type Ⅰ and(or) Ⅱ TZ (1.5%, 8/524; P<0.05). The proportion of HSIL detected by endocervical curettage alone was greater in post-menopausal patients (9.9%, 21/213) compared with pre-menopausal patients (2.6%, 32/1 236; P<0.05). (3)Initial treatment with LEEP: the positive rate of endocervical margin was significantly greater in post-menopausal patients (20.5%, 36/176) compared with pre-menopausal patients (10.5%, 130/1 236;P<0.05); in patients who were diagnosed as HSIL after LEEP, the positive rate of endocervical margin and the residual rate were both greater in post-menopausal patients compared with pre-menopausal patients [15.4% (25/162) versus 8.8% (105/1 189), P=0.008; 52.0% (13/25) versus 26.7% (28/105), P=0.014]. (4)Thirty-seven post-menopausal patients were treated by extrafascial hysterectomy as the primary therapy, 5 cases (13.5%, 5/37) were diagnosed as cervical cancer (stage Ⅰa1) after the surgery. Conclusions: (1) The lesions of HSIL in post-menopausal patients still have definite features under colposcopy as same as pre-menopausal patients. Endocervical curettage could help detect more HSIL in post-menopausal patients. Compared with pre-menopausal patients, post-menopausal HSIL patients have an increased risk of cervical cancer and are more likely missed by cervical tissue sampling. (2) LEEP has the dual effects of diagnosis and treatment, and is still the recommended treatment for post-menopausal HSIL patients. However, the increase in positive rate of endocervical margin and residual rate requires further active intervention. (3) Considering those post-menopausal HSIL patients who cannot accept conization as the initial treatment, the selection of hysterectomy type requires more thorough study.

[Analysis of the missed diagnosis of invasive carcinoma under the microscope in HSIL diagnosed by colposcopy-guided biopsy and related influencing factors].

Objective: To observe the missed diagnosis of invasive carcinoma under the microscope (ICUM) in high grade squamous intraepithelial neoplasia (HSIL) , and analyze associated factors influencing missed ICUM. Methods: A retrospective study was performed on patients diagnosed with HSIL by colposcopy-guided biopsy and treated with loop electrosurgical excision procedure (LEEP) at the First Affiliated Hospital of Nanjing Medical University, from December 2014 to December 2016. They were non-pregnant, ≤50 years old and the cervical volume without obvious enlargement and exogenous surface without and ulcerative lesions. A total of 283 cases with early cervical cytology results, never received cervical traumatic treatment or cervical biopsy in another hospital before, and their colposcopic images were clear enough to reevaluate. The ultimate pathological diagnosis was based on the higher-level pathological diagnosis between the results of cervical biopsy and LEEP to evaluate ICUM missed in HSIL and the risk factors. Results: (1) Among the 283 cases with HSIL diagnosed by colposcopy-directed biopsy, 44 cases (15.5%, 44/283) were missed diagnosis of ICUM, which consisted of 29 cases Ⅰ a1, 4 cases Ⅰ a2 and 11 cases Ⅰ b1 in the ultimate pathology. (2) Analysis of associated factors for missed ICUM: univariate analysis showed that, as the age increased, the risk of missed ICUM also increased (the rates of missed diagnosis for <30, 30-39, 40-50 years were 7.7%, 11.5%, 22.0%, respectively; χ(2)=6.254, P=0.012 by trend test) . The more the number of high-grade features, the higher risks (the rates of missed diagnosis for 1, 2, 3, 4 high-grade features were 10.2%, 17.6%, 23.8%, 30.8%, respectively; χ(2)=7.686, P=0.006 by trend test) . The locations of HSIL were only endocervical, only ectocervical and mixed, the risk increased by this sequence (2.8%, 5.1%, 28.7%; χ(2)=26.193, P<0.01 by trend test) . The rate of missed diagnosis for not completely visible squamocolumnar junction (SCJ) was higher than that of the completely visible one (22.3% vs 2.1%; χ(2)=19.680, P<0.01) . The rate of missed diagnosis was higher for existing atypical vessels than those without (60.7% vs 10.6%; χ(2)=48.279, P<0.01) . The rate of missed diagnosis for visible lesion size ≥40 mm(2) was higher than that of <40 mm(2) (27.3% vs 4.2%; χ(2)=28.921, P<0.01) . The rate of missed diagnosis for the proportion of visible lesion size in ectocervical size ≥0.75 was higher than that of <0.75 (83.3% vs 14.1%; P<0.01) . The rate of missed diagnosis for the maximum linear length of visible lesion ≥10 mm was higher than that of <10 mm (46.9% vs 9.0%; χ(2)=44.473, P<0.01) . But the different severity of cervical cytology before colposcopy was not associated with missed ICUM (P>0.05) . Multivariable analysis found that visibility of SCJ, atypical vessels, visible lesion size and maximum linear length of visible lesion were associated with missed diagnosis of ICUM (all P<0.05) . Conclusions: The diagnostic value of HSIL by colposcopy is limited. Meanwhile, for the patients who are ≤50 years old with HSIL diagnosed by cervical biopsy, invisibility of SCJ, atypical vessels, visible lesion size and maximum linear length of visible lesion evaluated by colposcopy are the independent risk factors of missed ICUM. Thereby, it is necessary to take active intervention for HSIL with these risk factors.

[Audiological basis of continuing to wear hearing aids in the contralateral ear of young children after cochlear implantation].

Objective: To study the accuracy of preoperative-ASSR in predicting behavioral thresholds of children at low age with profound hearing loss and to evaluate the feasibility of using hearing aids in non-operating ear after the cochlear implantation.Method: Children aged less than 2 years old at the time of surgery were selected as study subjects.The preoperative ASSR response threshold in 0.5, 1.0, 2.0 and 4.0 kHz were recorded.The play audiometry test was used to get the non operative ear behavioral thresholds of the children after three years old. Altogether 179 children with good cooperation and reliable results were enrolled in this study. The results of behavioral thresholds were made a comparison with the results of ASSR response threshold with corresponding frequency before operation for statistical analysis.Result: The behavioral threshold is better than corresponding ASSR response threshold with significant differences(P<0.01). Prediction rates of ASSR response threshold corresponding to behavioral thresholds from 0.5 kHz to 4.0 kHz are 49.7%, 70.9%, 59.2% and 60.9%.There are cases at all frequencies where ASSR showed no reaction but a behavioral thresholds were detected. The proportions of undrawn ASSR reaction at each frequency were 83.3%,75.5%,64.9%,45.1%.Conclusion: In profound hearing loss, the deviation between preoperative ASSR response thresholds and postoperative behavioral thresholds exists, especially in low frequency. No ASSR reaction in clinic cannot represent that there is no measurable hearing threshold. We suggest children at low age continue to wear hearing aids in the non operative ear after cochlear implantation. Whether to give up hearing aids should be judged after gaining the behavioral audiometry to avoid missing the bimodal opportunity.

[The long-term benefits of cochlear implantation for children and their families].

Objective:To study the auditory language ability of children with artificial cochlea after entering normal schools as well as the school life conditions and to evaluate the long-term comprehensive benefits of surgery on children along with their families. Method:The questionnaire was designed referring to the categories of auditory performance (CAP), the speech intelligibility rating (SIR), and family burden scale of diseases (FBS), with the questions involving four aspects of the general conditions, the auditory language ability, the school life conditions and the family conditions. Altogether 50 families were participated. Children who had applied artificial cochlea for at least two years and studied in normal schools were selected as the studying objects. The follow-up questionnaire was conducted on the parents. The differences were compared in the auditory language ability and the school life conditions, grouped by gender, age at surgery, parents' educational level, residence, service time of artificial cochlea and parent-child time in general. Meanwhile, the differences of 15 family conditions were compared before and after surgery. Result:The variable factors were of no obvious statistical significance in the comparison among groups of the auditory language ability (P>0.05); In the comparison of the school life conditions, there were significant differences in the number of making friends when grouped by parent-child time (P<0.05), there wes no statistical significance in the comparison among groups of the remaining various factors (P>0.05); In the comparison of family conditions before and after surgery, the rest of the indexes were of significant differences (P<0.05), there was no significant differences in the economic burden and social time of parents (P>0.05). Family conditions improved evidently after surgery. Conclusiont:Under the circumstances of different variable factors, after 2 years of rehabilitation, children are able to acquire better auditory language ability, and adjust themselves to the school life, finishing their studies, still there is a deficiency in the aspect of organizational competence in comparison to age-matched children. In addition, with children's recovery and attending schools, parents' mental stress is significantly reduced, family life returns to normal, and family atmosphere becomes harmonious. Not only can the artificial cochlea help rid children of disability, but also it can be of obvious benefits to their families.

Association between killer cell immunoglobulin-like receptor 2DS5 gene with essential hypertension in the Chinese Han patients.

Hypertension has been suggested to be mediated by immunity and inflammation. As immune system genes, killer cell immunoglobulin-like receptors (KIRs) genes play an important role in the pathogenesis of autoimmune diseases. We conducted a community population-based case-control study to analyse associations between KIR genes and hypertension. KIR genes were genotyped using sequence-specific primer polymerase chain reaction in 380 unrelated essential hypertensives and 527 normotensives. The frequencies of KIR2DS5 gene were significantly decreased in participants with high high-sensitivity C-reactive protein (hsCRP) compared with participants with low hsCRP (17.02% vs 34.08%, p = .016). The presence of KIR2DS5 gene was independently associated with decreased risk of hypertension (odds ratio = 0.62; 95% confidence interval: 0.43-0.89), after adjusted for conventional risk factors, hsCRP and other KIR genes. We found that participants with KIR2DS5 gene had a decreased risk of hypertension compared with participants without KIR2DS5 gene. Our findings suggested that KIR2DS5 gene may be protective against hypertension.

[Expression of E-cadherin, N-cadherin, ß-catenin and their clinical significance in laryngeal carcinoma].

OBJECTIVE: To investigate the expression of E-cadherin(E-cad), N-cadherin(N-cad), ß-catenin(ß-cat), which are the markers of Epithelial-mesenchymal transition and analyze their relationships with the clinicopathological features and the prognosis of the laryngeal squamous cell carcinoma. METHODS: The expression levels of E-cad, N-cad, ß-cat in 76 tumor tissues and their corresponding adjacent normal laryngeal tissues were determined by immunohistochemistry method. Relationships between the proteins' expression and clinicopathological features were analyzed. Survival curves were calculated using the Kaplan-Meier method. Differences in the survival rates were analyzed by the log-rank test among different expression groups. Cox's regression model was used to examine the independent predictor of the prognosis of the laryngeal cancer. RESULTS: The expression levels of E-cad, N-cad, ß-cat in tumor tissues and adjacent normal tissues were statistically significant(P<0.001). The expression level of E-cad and ß-cat in the laryngeal carcinoma was related to lymph node metastasis, clinical pathological stage and differentiation degree (P<0.05), while N-cad expression level was associated with clinical stage and differentiation degree (P<0.05). The expression of E-cad was correlated with the expression of ß-cat (P=0.001), and the expression of N-cad was correlated with ß-cat (P=0.02), but the expression of E-cad was not correlated with N-cad. There were four subgroups of patterns of E-cad and N-cad expression: E-cad (+ )/N-cad (-), E-cad (+ ) /N-cad (+ ), E-cad (-) /N-cad (-), E-cad (-) /N-cad (+ ). The expression level of each group was related to the clinical pathological stage and differentiation degree (P<0.05). The expression level of E-cad/ß-cat was associated with lymph node metastasis, clinical pathological stage and differentiation degree (P<0.01). Log-rank analysis showed that the prognosis of negative and positive groups was statistically different (P<0.05), and the combined analysis showed that the prognosis of E-cad/N-cad or E-cad/ß-cat group was significantly different (P<0.01). Cox's regression model analysis showed that the clinical stage and ß-cat were independent predictors of the prognosis of laryngeal carcinoma. CONCLUSIONS: The low expression of E-cad, high abnormal expression of N-cad and ß-cat played an important role in the occurrence and development of laryngeal carcinoma. It can provide a reference for evaluating clinical prognosis. The clinical pathological stage and ß-cat can be used as independent predictors for the prognosis of laryngeal carcinoma.

Long-range and high-precision correlation optical time-domain reflectometry utilizing an all-fiber chaotic source.

We propose a long range, high precision optical time domain reflectometry (OTDR) based on an all-fiber supercontinuum source. The source simply consists of a CW pump laser with moderate power and a section of fiber, which has a zero dispersion wavelength near the laser's central wavelength. Spectrum and time domain properties of the source are investigated, showing that the source has great capability in nonlinear optics, such as correlation OTDR due to its ultra-wide-band chaotic behavior, and mm-scale spatial resolution is demonstrated. Then we analyze the key factors limiting the operational range of such an OTDR, e. g., integral Rayleigh backscattering and the fiber loss, which degrades the optical signal to noise ratio at the receiver side, and then the guideline for counter-act such signal fading is discussed. Finally, we experimentally demonstrate a correlation OTDR with 100km sensing range and 8.2cm spatial resolution (1.2 million resolved points), as a verification of theoretical analysis.

Toward integration of comparative genetic, physical, diversity, and cytomolecular maps for grasses and grains, using the sorghum genome as a foundation.

The small genome of sorghum (Sorghum bicolor L. Moench.) provides an important template for study of closely related large-genome crops such as maize (Zea mays) and sugarcane (Saccharum spp.), and is a logical complement to distantly related rice (Oryza sativa) as a "grass genome model." Using a high-density RFLP map as a framework, a robust physical map of sorghum is being assembled by integrating hybridization and fingerprint data with comparative data from related taxa such as rice and using new methods to resolve genomic duplications into locus-specific groups. By taking advantage of allelic variation revealed by heterologous probes, the positions of corresponding loci on the wheat (Triticum aestivum), rice, maize, sugarcane, and Arabidopsis genomes are being interpolated on the sorghum physical map. Bacterial artificial chromosomes for the small genome of rice are shown to close several gaps in the sorghum contigs; the emerging rice physical map and assembled sequence will further accelerate progress. An important motivation for developing genomic tools is to relate molecular level variation to phenotypic diversity. "Diversity maps," which depict the levels and patterns of variation in different gene pools, shed light on relationships of allelic diversity with chromosome organization, and suggest possible locations of genomic regions that are under selection due to major gene effects (some of which may be revealed by quantitative trait locus mapping). Both physical maps and diversity maps suggest interesting features that may be integrally related to the chromosomal context of DNA-progress in cytology promises to provide a means to elucidate such relationships. We seek to provide a detailed picture of the structure, function, and evolution of the genome of sorghum and its relatives, together with molecular tools such as locus-specific sequence-tagged site DNA markers and bacterial artificial chromosome contigs that will have enduring value for many aspects of genome analysis.

New approach for inhibiting Rev function and HIV-1 production using the influenza virus NS1 protein.

The Rev protein of HIV-1, which facilitates the nuclear export of HIV-1 pre-mRNAs, has been a target for antiviral therapy. Here we describe a new strategy for inhibiting Rev function and HIV-1 replication. In contrast to previous approaches, we use a wild-type rather than a mutant Rev protein and covalently link this Rev sequence to the NS1 protein of influenza A virus, a protein that inhibits the nuclear export of mRNAs. The NS1 protein contains an RNA-binding domain mutation (RM), so that the only functional RNA-binding domain in the chimeric protein (NS1RM-Rev) is in the Rev protein sequence. In the presence of the NS1RM-Rev chimeric protein, HIV-1 pre-mRNAs were retained in, rather than exported from, the nucleus. In addition, this chimeric protein effectively inhibited Rev function in trans in transfection experiments and effectively inhibited the production of HIV-1 in tissue culture cells transfected with an infectious molecular clone of HIV-1 DNA. The inhibitory activities of the NS1RM-Rev chimera were at least equivalent to those of the Rev M10 mutant protein, which has been considered to be the prototype trans inhibitor of Rev function and is currently in phase I clinical trials for the treatment of AIDS patients. We discuss (i) the potential for increasing the inhibitory activity of NS1-Rev chimeras against HIV-1 and (ii) the need for additional studies to evaluate these chimeras for the treatment of AIDS.

An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure.

The NS1 protein of influenza A virus has the unique property of binding to three apparently different RNAs: poly A; a stem-bulge in U6 small nuclear RNA; and double-stranded RNA. One of our major goals is to determine how the NS1 protein recognizes and binds to its several RNA targets. As the first step for conducting structural studies, we have succeeded in identifying a fragment of the NS1 protein that possesses all the RNA-binding activities of the full-length protein. The RNA-binding fragment consists of the 73 amino-terminal amino acids of the protein. We have developed procedures for obtaining large amounts of the polypeptide in pure form. This has enabled us to establish the RNA-binding properties of this polypeptide and to demonstrate that it retains the ability to dimerize exhibited by the full-length protein. In addition, far-UV CD spectroscopy indicates that this RNA-binding polypeptide is largely (approximately 80%) helical, suggesting that the mode of dimerization of the NS1 protein and of its interaction with RNA is mediated, at least in part, by helices.

The influenza virus NS1 protein forms multimers in vitro and in vivo.

The NS1 protein of the influenza A virus inhibits both the nuclear export of mRNA and pre-mRNA splicing. Two functional domains, an RNA-binding domain and an effector domain, have been identified in this protein. Here we demonstrate that the NS1 protein exists as a dimer in vitro both in the absence of its RNA target and when it is bound to a specific RNA target, U6 snRNA. This indicates that it is most likely the dimer that binds to the RNA target. Mutational analysis indicated that the RNA-binding and dimerization domains are coincident. Multimerization also occurs in vivo, as assayed using the yeast two-hybrid system. In contrast to the situation in vitro, multimerization in vivo was mediated by not only the RNA-binding domain but also the effector domain. This suggests that multimerization in vivo involves a cellular protein cofactor that bridges more than one NS1 protein molecule together via their effector domains.

The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing.

We have shown previously that the influenza virus NS1 protein inhibits the nuclear export of mRNAs. Here we demonstrate that the NS1 protein also regulates another post-transcriptional step: It inhibits pre-mRNA splicing both in vivo and in vitro. The mode by which the NS1 protein inhibits pre-mRNA splicing is novel. The pre-mRNA forms spliceosomes, but subsequent catalytic steps in splicing are inhibited. Affinity selection experiments establish that the NS1 protein is associated with the spliceosomes that are formed. The RNA-binding domain of the NS1 protein is required for the inhibition of splicing and for the interaction of the protein with spliceosomes. Because the NS1 protein is associated with U6 snRNA in influenza virus-infected cells as well as in splicing extracts from uninfected cells, it is likely that the NS1 protein also inhibits pre-mRNA splicing in infected cells. Surprisingly, the splicing of the viral ns1 mRNA, the very mRNA that encodes the NS1 protein, was resistant to inhibition by the NS1 protein. This resistance is conferred by sequences in ns1 mRNA.

Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA.

The influenza virus NS1 protein is the only known example of a protein that inhibits the nuclear export of mRNA. To identify the functional domains of this protein, we introduced 18 2- or 3-amino-acid substitutions at approximately equally spaced locations along the entire length of the protein. Two functional domains were identified. The domain near the amino end (amino acids 19 through 38) was shown to be the RNA-binding domain, by using a gel shift assay with purified NS1 protein and spliced viral NS2 mRNA as the RNA target. The second domain, which is in the carboxy half of the molecule, was presumed to be the effector domain that interacts with host nuclear proteins to carry out the nuclear RNA export function, by analogy with the effector domain of the Rev proteins of human immunodeficiency virus (HIV) and other lentiviruses which facilitate rather than inhibit nuclear RNA export. The NS1 protein has a 10-amino-acid sequence that is similar to the consensus sequence in the effector domains of lentivirus Rev proteins, specifically including two crucial leucines at positions 7 and 9 of this sequence. However, the effector domains of the NS1 and Rev (HIV type 1 [HIV-1]) proteins differed in several significant ways including the following: (i) unlike the HIV-1 Rev protein, NS1 effector domain mutants were negative recessive rather than negative dominant, (ii) the NS1 effector domain is about three times larger than the effector domain of the HIV-1 Rev protein, and (iii) unlike the HIV-1 protein, NS1 effector domain mutants exhibited a surprising property, a changed intracellular/intranuclear distribution, compared with the wild-type protein. These differences strongly suggest that the effector domains of the NS1 and Rev proteins interact with different nuclear protein targets, which likely explains the opposite effects of these two proteins on nuclear mRNA export.

Nuclear matrix-intermediate filament system and its alteration in adenovirus infected HeLa cell.

The nuclear matrix-intermediate filament (NM-IF) of normal and adenovirus infected HeLa cells were investigated by means of both electron microscopy and two-dimensional gel electrophoresis. After infection there were some changes in NM-IF, and the viral factory was suspended in nuclear matrix. Two new polypeptides appeared in 2-D gel of NM-IF after infection. They are probably not viral proteins but cellular polypeptides. During viral replication, all of vimentin were degraded and phosphorylated keratin 18 increased. These facts suggest that NM-IF plays a certain role in adenovirus replication.