Rolling replication of short DNA circles.

Natural genes and proteins often contain tandemly repeated sequence motifs that dramatically increase physiological specificity and activity. Given the selective value of such repeats, it is likely that several different mechanisms have been responsible for their generation. One mechanism that has been shown to generate relatively long tandem repeats (in the kilobase range) is rolling circle replication. In this communication, we demonstrate that rolling circle synthesis in a simple enzymatic system can produce tandem repeats of monomers as short as 34 bp. In addition to suggesting possible origins for natural tandem repeats, these observations provide a facile means for constructing libraries of repeated motifs for use in "in vitro evolution" experiments designed to select molecules with defined biological or chemical properties.     

Patterns of known and novel small RNAs in human cervical cancer

Recent studies suggest that knowledge of differential expression of microRNAs (miRNA) in cancer may have substantial diagnostic and prognostic value. Here, we use a direct sequencing method to characterize the profiles of miRNAs and other small RNA segments for six human cervical carcinoma cell lines and five normal cervical samples. Of 166 miRNAs expressed in normal cervix and cancer cell lines, we observed significant expression variation of six miRNAs between the two groups. To further show the biological relevance of our findings, we examined the expression level of two significantly varying miRNAs in a panel of 29 matched pairs of human cervical cancer and normal cervical samples. Reduced expression of miR-143 and increased expression of miR-21 were reproducibly displayed in cancer samples, suggesting the potential value of these miRNAs as tumor markers. In addition to the known miRNAs, we found a number of novel miRNAs and an additional set of small RNAs that do not meet miRNA criteria.     

Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated

The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non-HIV-1 antigens.     

多西他赛联合顺铂治疗晚期乳腺癌30例

目的观察多西他赛联合顺铂方案治疗晚期乳腺癌患者的临床疗效及引起的不良反应。方法对郑州市消防支队卫生队收治的30例经病理组织学确诊的晚期乳腺癌患者进行多西他赛35mg/m2,静脉滴注1h,第1、8、15d,化疗前1d给予地塞米松每次7.5mg,每日2次,连用3d。顺铂20mg/m2,静脉滴注第1～5d。21d为1个周期,至少应用2个周期后按照WHO标准进行疗效评价。结果晚期乳腺癌30例中完全缓解(CR)4例,部分缓解(PR)15例,稳定(SD)7例,进展(PD)4例,总有效率(PR+CR)为63%,无严重不良反应。结论多西他赛联合顺铂治疗晚期乳腺癌疗效确切,不良反应可以耐受,临床可广泛推广。     

MicroRNA expression signature of human sarcomas

MicroRNAs (miRNAs) are approximately 22 nucleotide-long noncoding RNAs involved in several biological processes including development, differentiation and proliferation. Recent studies suggest that knowledge of miRNA expression patterns in cancer may have substantial value for diagnostic and prognostic determinations as well as for eventual therapeutic intervention. We performed comprehensive analysis of miRNA expression profiles of 27 sarcomas, 5 normal smooth muscle and 2 normal skeletal muscle tissues using microarray technology and/or small RNA cloning approaches. The miRNA expression profiles are distinct among the tumor types as demonstrated by an unsupervised hierarchical clustering, and unique miRNA expression signatures were identified in each tumor class. Remarkably, the miRNA expression patterns suggested that two of the sarcomas had been misdiagnosed and this was confirmed by reevaluation of the tumors using histopathologic and molecular analyses. Using the cloning approach, we also identified 31 novel miRNAs or other small RNA effectors in the sarcomas and normal skeletal muscle tissues examined. Our data show that different histological types of sarcoma have distinct miRNA expression patterns, reflecting the apparent lineage and differentiation status of the tumors. The identification of unique miRNA signatures in each tumor type may indicate their role in tumorigenesis and may aid in diagnosis of soft tissue sarcomas.     

High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment

The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.