Complete miRNA-15/16 loss in mice promotes hematopoietic progenitor expansion and a myeloid-biased hyperproliferative state.

Dysregulated apoptosis and proliferation are fundamental properties of cancer, and microRNAs (miRNA) are critical regulators of these processes. Loss of miR-15a/16-1 at chromosome 13q14 is the most common genomic aberration in chronic lymphocytic leukemia (CLL). Correspondingly, the deletion of either murine miR-15a/16-1 or miR-15b/16-2 locus in mice is linked to B cell lymphoproliferative malignancies. However, unexpectedly, when both miR-15/16 clusters are eliminated, most double knockout (DKO) mice develop acute myeloid leukemia (AML). Moreover, in patients with CLL, significantly reduced expression of miR-15a, miR-15b, and miR-16 associates with progression of myelodysplastic syndrome to AML, as well as blast crisis in chronic myeloid leukemia. Thus, the miR-15/16 clusters have a biological relevance for myeloid neoplasms. Here, we demonstrate that the myeloproliferative phenotype in DKO mice correlates with an increase of hematopoietic stem and progenitor cells (HSPC) early in life. Using single-cell transcriptomic analyses, we presented the molecular underpinning of increased myeloid output in the HSPC of DKO mice with gene signatures suggestive of dysregulated hematopoiesis, metabolic activities, and cell cycle stages. Functionally, we found that multipotent progenitors (MPP) of DKO mice have increased self-renewing capacities and give rise to significantly more progeny in the granulocytic compartment. Moreover, a unique transcriptomic signature of DKO MPP correlates with poor outcome in patients with AML. Together, these data point to a unique regulatory role for miR-15/16 during the early stages of hematopoiesis and to a potentially useful biomarker for the pathogenesis of myeloid neoplasms.

Genome-Wide Identification and Expression Analysis of Fatty Acid Desaturase (FAD) Genes in Camelina sativa (L.) Crantz.

Camelina sativa (L.) Crantz is an indispensable oilseed crop, and its seeds contain many unsaturated fatty acids. FAD (fatty acid desaturase) regulates the synthesis of unsaturated fatty acids. In this research, we performed CsFAD gene family analysis and identified 24 CsFAD genes in Camelina, which were unevenly distributed on 14 of the 19 total chromosomes. Phylogenetic analysis showed that CsFAD includes four subfamilies, supported by the conserved structures and motifs of CsFAD genes. In addition, we investigated the expression patterns of the FAD family in the different tissues of Camelina. We found that CsFAD family genes were all expressed in the stem, and CsFAD2-2 was highly expressed in the early stage of seed development. Moreover, during low temperature (4 °C) stress, we identified that the expression level of CsFAD2-2 significantly changed. By observing the transient expression of CsFAD2-2 in Arabidopsis protoplasts, we found that CsFAD2-2 was located on the nucleus. Through the detection and analysis of fatty acids, we prove that CsFAD2-2 is involved in the synthesis of linolenic acid (C18:3). In conclusion, we identified CsFAD2-2 through the phylogenetic analysis of the CsFAD gene family and further determined the fatty acid content to find that CsFAD2-2 is involved in fatty acid synthesis in Camelina.

Integration of sRNA, degradome, transcriptome analysis and functional investigation reveals gma-miR398c negatively regulates drought tolerance via GmCSDs and GmCCS in transgenic Arabidopsis and soybean.

BACKGROUND: Drought conditions adversely affect soybean growth, resulting in severe yield losses worldwide. Increasing experimental evidence indicates miRNAs are important post-transcriptional regulators of gene expression. However, the drought-responsive molecular mechanism underlying miRNA-mRNA interactions remains largely uncharacterized in soybean. Meanwhile, the miRNA-regulated drought response pathways based on multi-omics approaches remain elusive. RESULTS: We combined sRNA, transcriptome and degradome sequencing to elucidate the complex regulatory mechanism mediating soybean drought resistance. One-thousand transcripts from 384 target genes of 365 miRNAs, which were enriched in the peroxisome, were validated by degradome-seq. An integrated analysis showed 42 miRNA-target pairs exhibited inversely related expression profiles. Among these pairs, a strong induction of gma-miR398c as a major gene negatively regulates multiple peroxisome-related genes (GmCSD1a/b, GmCSD2a/b/c and GmCCS). Meanwhile, we detected that alternative splicing of GmCSD1a/b might affect soybean drought tolerance by bypassing gma-miR398c regulation. Overexpressing gma-miR398c in Arabidopsis thaliana L. resulted in decreased percentage germination, increased leaf water loss, and reduced survival under water deficiency, which displayed sensitivity to drought during seed germination and seedling growth. Furthermore, overexpressing gma-miR398c in soybean decreased GmCSD1a/b, GmCSD2a/b/c and GmCCS expression, which weakened the ability to scavenge O2.-, resulting in increased relative electrolyte leakage and stomatal opening compared with knockout miR398c and wild-type soybean under drought conditions. CONCLUSION: The study indicates that gma-miR398c negatively regulates soybean drought tolerance, and provides novel insights useful for breeding programs to improve drought resistance by CRISPR technology.

An iron rheostat controls hematopoietic stem cell fate.

Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.

The corrosion resistance of composite arch wire laser-welded by NiTi shape memory alloy and stainless steel wires with Cu interlayer in artificial saliva with protein.

In this paper, the corrosion resistance of laser-welded composite arch wire (CoAW) with Cu interlayer between NiTi shape memory alloy and stainless steel wire in artificial saliva with different concentrations of protein was studied. It was found that protein addition had a significant influence on the corrosion behavior of CoAW. Low concentration of protein caused the corrosion resistance of CoAW decrease in electrochemical corrosion and immersion corrosion tests. High concentration of protein could reduce this effect.

Computed tomography-guided localization of pulmonary nodules prior to thoracoscopic surgery.

With the increasing awareness of physical examination, the detection rate of pulmonary nodules is gradually increasing. For pulmonary nodules recommended for management by video-assisted thoracic surgery (VATS), preoperative localization of the nodule is required if its location is difficult to determine intraoperatively by palpation. The computed tomography (CT)-guided preoperative localization technique is the most widely used method with low operational difficulty and high efficiency, which can include hook wire, microcoil, medical dye, medical surgical adhesive, combined application, and emerging localization techniques according to the material classification. Each method has its corresponding advantages and disadvantages, but there is still a lack of unified guidelines or standards for the selection of CT-guided preoperative localization methods in clinical practice. This review summarizes the operation precautions, advantages, and shortcomings of the above localization techniques in order to provide references for clinical application.

Colitis induced by PD-1 inhibitor combined with platinum-containing dual drug chemotherapy in Lewis mice and its mechanism.

Aims: To explore the occurrence and possible mechanism of colitis in Lewis mice treated with PD-1 inhibitor combined with platinum-containing dual drug chemotherapy. Subjects and Methods: A Lewis lung cancer model of C57BL/6 mice was established, randomly divided into the treatment group (group C, PD-1 inhibitor + Carboplatin (CARB) + Pemetrexed (PEM)) and model group (group B, normal saline), and a control group (group A, normal saline) was set up. Observe the changes in tumor-free weight, tumor volume, disease activity index (DAI), colon histopathology, identify serum interleukin (IL)-10, interferon (IFN)-γ, the expression of claudin-1, and occludin mRNA in the colon in each animals. Results: Compared with group A, the tumor-free weight of mice in B decreased (P < 0.001), the content of IL-10 in serum increased (P < 0.01), the content of IFN-γ in serum decreased (P < 0.01). Compared with group B, the transplanted tumor volume in C was reduced (P < 0.05), DAI scores of D4 (P < 0.001), and D7 (P < 0.001) were increased, colonic histopathology analysis showed that colitis occurred, serum IL-10 content was decreased (P < 0.05), IFN-γ content was increased (P < 0.05), and the mRNA expression of claudin-1 (P < 0.05) and occludin (P < 0.05) was reduced. Conclusions: This treatment can inhibit the growth of transplanted tumors but will cause colitis in Lewis mice. The impairment of intestinal barrier function following administration cause an imbalance in the expression of pro-inflammatory and anti-inflammatory factors in the colon, thus causing colitis.

Preparation of High Conductive Medium and Establishment of Laege Capacity Conductive Channel.

The preparation of highly conductive media and the construction of conducting channels play a crucial role in improving the electrical conductivity of electrically conductive adhesives. Therefore, a new MXene structure is reported in this paper, and the improved structure is rationally designed by computational modeling, which greatly prevents the buildup of MXene nanosheets, improves the stability of the structure, and creates a wide electron transfer channel, and the capacitance contribution of this structure is up to 86.3%. By mixing MXene modified with Ag-plated copper powder in a quantitative relationship to form high conductive media, the electrical conductivity is largely improved and the defect of low electron transfer rate of conventional conductive fillers is broken. The potential value of high conductive media is largely exploited using high throughput and machine learning methods, and here we show that the resistivity has reached 9.668 × 10-7 Ω m. The first principles investigate the conductive channels and electron transfer pathways of high-conductive media at the atomic level, further revealing the mechanism of action of high-conductive media. This study is also the first report on the application of MXene to high-conductive media.

Factors that contribute to false-negative results in CT-guided transthoracic lung core-needle biopsy.

Objective: To investigate the possibility of false-negative occurrence of non-specific benign pathological results on CT-guided transthoracic lung core-needle biopsy and identify risk factors for false-negative results. Methods: The clinical, imaging, and surgical data of 403 lung biopsy patients were retrospectively analyzed. Patients were divided into true-negative and false-negative (FN) groups according to the final diagnosis. Univariate analysis was used to compare the variables in two groups for statistical differences, and multivariate analysis was used to clarify the risk factors associated with FN results. Results: Of the 403 lesions, 332 were finally confirmed as benign and 71 to be malignant, with a FN rate of 17.6%. Older patient age (P = 0.01), burr sign (P = 0.00), and pleural traction sign (P = 0.02) were independent risk factors for FN results. The area under the receiver operating characteristic (ROC) curve's area under curve (AUC) was 0.73. Conclusion: CT-guided transthoracic lung core-needle biopsy has a high diagnostic accuracy and low rate of FN results. Older patient age, the burr sign, and the pleural traction sign are independent risk factors for FN results that should be monitored prior to surgery to reduce the risk of FN results.

Preoperative sarcopenia and systemic immune-inflammation index can predict response to intravesical Bacillus Calmette-Guerin instillation in patients with non-muscle invasive bladder cancer.

Background: To explore the prognostic significance of sarcopenia and systemic immune-inflammation index (SII) for response to intravesical Bacillus Calmette-Guerin (BCG) in patients with intermediate-, and high-risk non-muscle invasive bladder cancer (NMIBC). Methods: We retrospectively analyzed 183 consecutive patients treated in Qilu hospital of Shandong University for a first diagnosis of intermediate and high risk NMIBC. Using computed tomography scans at the third lumbar vertebra level, we calculated skeletal muscle index (SMI). Sarcopenia was defined as SMI <43 cm2/m2 for males with BMI < 25 kg/m2, <53 cm2/m2 for males with BMI ≥ 25 kg/m2, and <41 cm2/m2 for females. The response to intravesical BCG immunotherapy and relapse-free survival (RFS) were analyzed. Results: Compared with BCG responders, BCG non-responders were associated with sarcopenia (P < 0.001), carcinoma in situ (P < 0.001), T1 stage (P < 0.001), multiple tumor (P < 0.001), tumor diameter >=3cm (P < 0.001), and have a significant increase of neutrophil-to-lymphocyte ratio (NLR) (P < 0.001), platelet to lymphocyte ratio (PLR) (P = 0.004), SII (P < 0.001). The area under the ROC curve (AUC) of the BMI, NLR, PLR, and SII for response to intravesical BCG immunotherapy were 0.425, 0.693, 0.631, and 0.702 respectively. Logistic regression analysis demonstrated that sarcopenia and SII were predictors of response to intravesical BCG immunotherapy. The Kaplan-Meier survival analysis showed that the RFS of patients with BCG response, lower SII and no sarcopenia was significantly increased compared with that of patients with BCG non-response, higher SII and sarcopenia, respectively. Subgroup analysis demonstrated that the RFS of patients with high SII and sarcopenia was significantly decreased compared with those with low SII and no sarcopenia in Ta stage subgroup, T1 stage subgroup, non-Cis subgroup, multiple tumor subgroup, single tumor subgroup, tumor diameter≥3cm subgroup and tumor diameter<3cm subgroup, respectively (P < 0.05). However, there was no significant difference in RFS for patients in CIS subgroup (P > 0.05). Multivariate Cox analysis shown that sarcopenia (p=0.005) and high SII (p = 0.003) were significantly associated with poor RFS. Conclusions: Both sarcopenia and high SII are useful predictors of response to intravesical BCG in intermediate- and high-risk NMIBC patients. Patients with intermediate- and high-risk NMIBC that had sarcopenia or high SII at diagnosis were associated with poor RFS, and the combination of sarcopenia and SII may be a better predictor of RFS.

Compound connection mechanism of Al2O3 ceramic and TC4 Ti alloy with different joining modes.

In this paper, laser welding-brazing of TC4 Titanium (Ti) alloy and Al2O3 ceramic dissimilar material was carried. The results showed that the Ti alloy and Al2O3 were joined by melting filler metal when the laser was concentrated in the Ti alloy side of the joint. The joint with one fusion weld and one brazed weld separated by remaining unmelted Ti alloy. Laser beam offset the Ti alloy 1.5 mm, Ti alloy would not be completely melted in joint. Through heat conduction, the filler metal melted occurred at the Ti-ceramic interface. A brazed weld was formed at the Ti-ceramic interface with the main microstructure of ß-CuZn + Ti2Zn3, ß-CuZn and Al2Cu + ß-CuZn. The joint fractured at the brazed weld with the maximum tensile strength of 169 MPa.

Genome-wide identification of soybean Shaker K+ channel gene family and functional characterization of GmAKT1 in transgenic Arabidopsis thaliana under salt and drought stress.

Potassium is a major cationic nutrient involved in numerous physiological processes in plants. The uptake of K+ is mediated by K+ channels and transporters, and the Shaker K+ channel gene family plays an essential role in K+ uptake and stress resistance in plants. However, little is known regarding this family in soybean. In this study, 14 members of the Shaker K+ channel gene family were identified in soybean and were classified into five groups. Protein domain analysis revealed that Shaker K+ channel gene members have an ion transport domain (ion trans), a cyclic nucleotide-binding domain, ankyrin repeat domains, and a dimerization domain in the potassium ion channel. Quantitative real-time polymerase chain reaction analysis indicated that the expression of eight genes (notably GmAKT1) in soybean leaves and roots was significantly increased in response to salt and drought stress. Furthermore, the overexpression of GmAKT1 in Arabidopsis enhanced root length, K+ concentration, and fresh/dry weight ratio compared with wild-type plants subjected to salt and drought stress; this suggests that GmAKT1 improves the tolerance of soybean to abiotic stress. Our results provide important insight into the characterization of Shaker K+ channel gene family members in soybean and highlight the function of GmAKT1 in soybean plants under salt and drought stress.

A soybean calcineurin B-like protein-interacting protein kinase, GmPKS4, regulates plant responses to salt and alkali stresses.

Calcineurin B-like protein-interacting protein kinases (CIPKs) are key elements of plant abiotic stress signaling pathways. CIPKs are SOS2 (Salt Overly Sensitive 2)-like proteins (protein kinase S [PKS] proteins) which all contain a putative FISL motif. It seems that the FISL motif is found only in the SOS2 subfamily of protein kinases. In this study, the full-length cDNA of a soybean CIPK gene (GmPKS4) was isolated and was revealed to have an important role in abiotic stress responses. A qRT-PCR analysis indicated that GmPKS4 expression is upregulated under saline conditions or when exposed to alkali, salt-alkali, drought, or abscisic acid (ABA). A subcellular localization assay revealed the presence of GmPKS4 in the nucleus and cytoplasm. Further studies on the GmPKS4 promoter suggested it affects soybean resistance to various stresses. Transgenic Arabidopsis thaliana and soybean hairy roots overexpressing GmPKS4 had increased proline content as well as high antioxidant enzyme activities but decreased malondialdehyde levels following salt and salt-alkali stress treatments. Additionally, GmPKS4 overexpression activated reactive oxygen species scavenging systems, thereby minimizing damages due to oxidative and osmotic stresses. Moreover, upregulated stress-related gene expression levels were detected in lines overexpressing GmPKS4 under stress conditions. In conclusion, GmPKS4 improves soybean tolerance to salt and salt-alkali stresses. The overexpression of GmPKS4 enhances the scavenging of reactive oxygen species, osmolyte synthesis, and the transcriptional regulation of stress-related genes.

MDMX acts as a pervasive preleukemic-to-acute myeloid leukemia transition mechanism.

MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/ß-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of ß-Catenin in a p53-independent manner. Wnt/ß-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/ß-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/ß-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Microglial Homeostasis Requires Balanced CSF-1/CSF-2 Receptor Signaling.

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r+/- mouse model of ALSP hypothesized a central role of elevated cerebral Csf2 expression. Here, we show that monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r+/- mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in humans. Our data provide insights into the mechanisms underlying ALSP. Because increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions.

Myelodysplastic syndrome progression to acute myeloid leukemia at the stem cell level.

Myelodysplastic syndromes (MDS) frequently progress to acute myeloid leukemia (AML); however, the cells leading to malignant transformation have not been directly elucidated. As progression of MDS to AML in humans provides a biological system to determine the cellular origins and mechanisms of neoplastic transformation, we studied highly fractionated stem cell populations in longitudinal samples of patients with MDS who progressed to AML. Targeted deep sequencing combined with single-cell sequencing of sorted cell populations revealed that stem cells at the MDS stage, including immunophenotypically and functionally defined pre-MDS stem cells (pre-MDS-SC), had a significantly higher subclonal complexity compared to blast cells and contained a large number of aging-related variants. Single-cell targeted resequencing of highly fractionated stem cells revealed a pattern of nonlinear, parallel clonal evolution, with distinct subclones within pre-MDS-SC and MDS-SC contributing to generation of MDS blasts or progression to AML, respectively. Furthermore, phenotypically aberrant stem cell clones expanded during transformation and stem cell subclones that were not detectable in MDS blasts became dominant upon AML progression. These results reveal a crucial role of diverse stem cell compartments during MDS progression to AML and have implications for current bulk cell-focused precision oncology approaches, both in MDS and possibly other cancers that evolve from premalignant conditions, that may miss pre-existing rare aberrant stem cells that drive disease progression and leukemic transformation.

Publisher Correction: Myelodysplastic syndrome progression to acute myeloid leukemia at the stem cell level.

In the version of this article originally published, Ulrich Steidl's name was listed as "and Ulrich Steidl." His name has been updated to "Ulrich Steidl." The error has been fixed in the print, PDF and HTML versions of this article.

Inactivation of p27Kip1 promotes chemical mouse liver tumorigenesis in the resistant strain C57BL/6J.

The biochemical function of p27Kip1 as an inhibitor of cyclin-dependent kinases is well-established, but the role of p27 as a tumor suppressor depends on specific cellular contexts. Previous studies using p27 knockout mice on mixed C57BL/6J x 129/Sv strain background did not find a tumor suppressor role of p27 in the liver. An important feature of mouse liver tumorigenesis is strain-dependent tumor susceptibility. Here, we determined the role of p27 in liver tumorigenesis in C57BL/6J mice, a liver tumor resistant strain, in response to a diethylnitrosamine (DEN) and phenolbarbital (PB) two-stage carcinogenesis protocol. At 6 mo of age, while livers of DEN-PB treated p27+/+ and p27-/- C57BL/6J mice appeared morphologically normal, p27-/- livers, but not p27+/+ livers, contained readily detectable glucose-6-phosphatase (G6Pase)-deficient foci. At the 9-mo time point, p27-/- mice developed significantly enhanced liver tumor phenotypes than p27+/+ mice as demonstrated by increased numbers and sizes of liver surface nodules, increased liver-to-body weight ratios, and increased numbers of G6Pase-deficient nodules and histologically diagnosed foci and adenomas in liver sections. Hepatic lesions in p27-/- livers contained more proliferating hepatocytes than lesions in p27+/+ livers, while the numbers of apoptotic cells appeared similar in lesions of both genotypes. Unexpectedly, tumors in p27-/- livers contained only slightly elevated Cdk2 kinase activity compared with normal livers. These results reveal a liver tumor suppressor role of p27 in this resistant mouse strain, and the need to further study the role of Cdk2 kinase in liver tumor promotion by p27 inactivation.

Disrupting Skp2-cyclin A interaction with a blocking peptide induces selective cancer cell killing.

Skp2 fulfills the definition of an oncoprotein with its frequent overexpression in cancer cells and oncogenic activity in various laboratory assays and therefore is a potential cancer therapy target. The best-known function of Skp2 is that of an F-box protein of the SCF(Skp2)-Roc1 E3 ubiquitin ligase targeting the cyclin-dependent kinase inhibitor p27(Kip1). Knockdown of Skp2 generally leads to accumulation of p27 but its effects on cancer cells are less certain. Another function of Skp2 is its stable interaction with cyclin A, which directly protects cyclin A from inhibition by p27 in in vitro kinase assays. Here, we report that an 18-residue blocking peptide of Skp2-cyclin A interaction can indirectly inhibit cyclin A/Cdk2 kinase activity dependent on the presence of p27 in in vitro kinase assays. Transmembrane delivery of this blocking peptide can induce cell death in a panel of four cancer cell lines in which Skp2 knockdown only have mild inhibitory effects. This Skp2-cyclin A interaction blocking peptide can synergize with a previously identified E2F1-derived LDL peptide, which blocks its access to cyclin A, in killing cancer cells. IC(50) of the Skp2-cyclin A blocking peptide correlated with abundance of Skp2, its intended target, in cancer cells. These results suggest that Skp2-cyclin A interaction plays an important role in cancer cell survival and is an attractive target for cancer drug discovery.

Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown.

A number of natural microRNA (miRNA) hairpins have been found in clusters of multiple identical or different copies, suggesting that effects of miRNAs can be enhanced and multiple genes can be regulated together by encoding multiple miRNA hairpins in a single transcript. Here, we report a simple and effective artificial multi-hairpin method that stimulates production of mature 22-nucleotide small RNAs from modified miRNA hairpins, improves gene knockdown over single-hairpin constructs, and provides linked multi-gene knockdowns.