Anatomic double bundle ACL reconstruction outperforms any types of single bundle ACL reconstructions in controlling dynamic rotational laxity.

PURPOSE: To compare the different types of ACL reconstructions in terms of knee dynamic laxity evaluated by acceleration. METHODS: Sixteen fresh frozen cadaveric knees were used. Pivot shift test was manually performed while monitoring the tibial acceleration by use of a triaxial accelerometer. The test was repeated before and after the ACL resection and reconstruction. Three types of ACL reconstruction were tested: (1) Anatomic Single-Bundle reconstruction (n = 8), the graft was placed at the center of the ACL footprint for the both femoral and tibial sides (tunnel diameter: 8mm); (2) Conventional Single-Bundle reconstruction (n = 8), the graft was placed from the tibial PL footprint to femoral high AM position (tunnel diameter: 8mm) and (3) Anatomic Double-Bundle reconstruction (n = 8). The acceleration in each of three x-y-z directions and the overall magnitude of acceleration was calculated to evaluate dynamic rotational laxity and compared between different ACL reconstructions. RESULTS: The overall magnitude of acceleration was significantly different between ACL intact and deficient knees (p < 0.0001). The acceleration was reduced by the DB ACL reconstruction to the intact level (n.s.), but the two SB ACL reconstruction failed to achieve the intact level of the acceleration (p = 0.0002non-anatomic SB, p < 0.0001 anatomic SB). CONCLUSION: The anatomic DB reconstruction better restores dynamic rotational laxity when compared to the SB ACL reconstructions no matter if the tunnel placement was anatomic. The anatomic DB reconstruction better restores dynamic rotational laxity when compared to both anatomic and non-anatomic SB ACL reconstruction. For this reason anatomic DB ACL reconstruction is recommended for cases where rotational laxity is an issue.

Effects of acute, intermittent exercise in hypoxic environments on the release of cardiac troponin.

The purpose of this study was to examine the effects of acute, intermittent exercise performed in hypoxic environments on the release of cardiac troponin (cTn). Ten well-trained, male marathon runners (22.1 ± 2.6 years, 64.0 ± 4.9 kg and 177.3 ± 3.9 cm) completed three intermittent exercise protocols under normoxic (trial N) and hypoxic (trial AH and RH) conditions. In trial N, the fraction of inspiration oxygen (FIO2 ) was 21.0% and exercise intensity was 90% and 50% normoxic velocity of VO2max (vVO2max). In trial AH, FIO2 was 14.4% (simulated altitude of 3000 m) and exercise intensity was 90% and 50% normoxic vVO2max. In trial RH, FIO2 was 14.4% and exercise intensity was 90% and 50% hypoxic vVO2max. High-sensitivity cardiac troponin T (hs-cTnT) and cardiac troponin I (cTnI) were measured pre- and 0, 2, 4, and 24 h post-exercise. Hs-cTnT was elevated in all three trials, peaking at 2 to 4 h and returning to the baseline 24 h post-exercise. CTnI increased in trial AH, peaking at 2 to 4 h and returning below the detection limit 24 h post-exercise. It is concluded that the stimulus of hypoxia did not in and of itself induce more cTn to be released, but exercise intensity could affect this response in hypoxic environments.

Is the native ACL insertion site "completely restored" using an individualized approach to single-bundle ACL-R?

PURPOSE: The goal of individualized anatomic anterior cruciate ligament reconstruction (ACL-R) is to reproduce each patient's native insertion site as closely as possible. The amount of the native insertion site that is recreated by the tunnel aperture area is currently unknown, as are the implications of the degree of coverage. As such, the goals of this study are to determine whether individualized anatomic ACL-R techniques can maximally fill the native insertion site and to attempt to establish a crude measure to evaluate the percentage of reconstructed area as a first step towards elucidating the implications of complete footprint restoration. METHODS: This is a prospective pilot study of 45 patients who underwent primary single-bundle anatomic ACL-R from May 2011 to April 2012. Length and width of the native insertion site were measured intraoperatively. Using published guidelines, reconstruction technique and graft choice were determined to maximize the percentage of reconstructed area. Native femoral and tibial insertion site area and femoral tunnel aperture area were calculated using the formula for area of an ellipse. On the tibial side, tunnel aperture area was calculated with respect to drill diameter and drill guide angle. Percentage of reconstructed area was calculated by dividing total tunnel aperture area by the native insertion site area. RESULTS: The mean areas for the native femoral and tibial insertion sites were 83 ± 20 and 125 ± 20 mm(2), respectively. The mean tunnel aperture area for the femoral side was 65 ± 17, and 86 ± 17 mm(2) for the tibial tunnel aperture area. On average, percentage of reconstructed area was 79 ± 13 % for the femoral side, and 70 ± 12 % for the tibial side. CONCLUSION: Anatomic ACL-R does not restore the native insertion site in its entirety. Percentage of reconstructed area serves as a rudimentary tool for evaluating the degree of native insertion site coverage using current individualized anatomic techniques and provides a starting point from which to evaluate the clinical significance of complete footprint restoration. LEVEL OF EVIDENCE: IV.

Anatomic anterior cruciate ligament (ACL) reconstruction: a global perspective. Part 1.

PURPOSE: In August 2011, orthopaedic surgeons from more than 20 countries attended a summit on anatomic anterior cruciate ligament (ACL) reconstruction. The summit offered a unique opportunity to discuss current concepts, approaches, and techniques in the field of ACL reconstruction among leading surgeons in the field. METHODS: Five panels (with 36 panellists) were conducted on key issues in ACL surgery: anatomic ACL reconstruction, rehabilitation and return to activity following anatomic ACL reconstruction, failure after ACL reconstruction, revision anatomic ACL reconstruction, and partial ACL injuries and ACL augmentation. Panellists' responses were secondarily collected using an online survey. RESULTS: Thirty-six panellists (35 surgeons and 1 physical therapist) sat on at least one panel. Of the 35 surgeons surveyed, 22 reported performing "anatomic" ACL reconstructions. The preferred graft choice was hamstring tendon autograft (53.1 %) followed by bone-patellar tendon-bone autograft (22.8 %), allograft (13.5 %), and quadriceps tendon autograft (10.6 %). Patients generally returned to play after an average of 6 months, with return to full competition after an average of 8 months. ACL reconstruction "failure" was defined by 12 surgeons as instability and pathological laxity on examination, a need for revision, and/or evidence of tear on magnetic resonance imaging. The average percentage of patients meeting the criteria for "failure" was 8.2 %. CONCLUSIONS: These data summarize the results of five panels on anatomic ACL reconstruction. The most popular graft choice among surgeons for primary ACL reconstructions is hamstring tendon autograft, with allograft being used most frequently employed in revision cases. Nearly half of the surgeons surveyed performed both single- and double-bundle ACL reconstructions depending on certain criteria. Regardless of the technique regularly employed, there was unanimous support among surgeons for the use of "anatomic" reconstructions using bony and soft tissue remnant landmarks.

The concept of individualized anatomic anterior cruciate ligament (ACL) reconstruction.

PURPOSE: To describe the concept of individualized anatomic anterior cruciate ligament (ACL) reconstruction. METHODS: The PubMed/Medline database was searched using keywords pertaining to ACL reconstruction. Relevant articles were reviewed in order to summarize important concepts of individualized surgery in ACL reconstruction. Surgical experiences with case examples are also highlighted. RESULTS: Individualized ACL surgery allows for the customization of surgery to each individual patient. Accounting for graft selection and other characteristics such as anatomy, lifestyle and activity preferences may provide the patient with the best potential for a successful outcome. The surgeon should be comfortable with a variety of graft harvests and surgical techniques when practicing individualized surgery. CONCLUSION: Individualized anatomic ACL reconstruction is founded on the objective evaluation of functional anatomy and individual characteristics, thereby restoring the ACL as closely as possible to the native anatomy and function. The adoption and subsequent use of individualized surgery may facilitate improved clinical as well as objective outcomes, particularly in the long term. LEVEL OF EVIDENCE: V.

Effect of ACL reconstruction tunnels on stress in the distal femur.

PURPOSE: This study examined the change in femoral stress caused by graft tunnels drilled for anterior cruciate ligament (ACL) reconstruction. Using a computational model, the number, geometry and position of the graft tunnels exits were varied to determine the effect on bone stress. METHODS: A finite element model of the distal femur was developed from a CT scan of a cadaveric knee. To assess the model, the strain calculated computationally was compared to experimentally measured strains in eleven unpaired human cadaver femurs. Using the computational model, the number, geometry and position of the graft tunnel exits were varied to determine the effect on bone stress based on the stress concentration factor: the ratio of bone stress with tunnels to intact bone stress. RESULTS: The results indicated that the second tunnel in double-bundle ACL reconstruction results in approximately a 20 % increase in the maximum femoral stress as compared to single-bundle reconstruction. The highest stresses occur at the tunnel exits. The position of the tunnel exits effects femoral stress with the stress increasing slightly (AM SCR from 0.7 to 1 and PL SCR from 1.2 to 1.3) when the AM tunnel exit is moved anteriorly and having greater increases as the posterior lateral (PL) tunnel exit is moved laterally (PL SCR from 1.2 to 1.7) or posteriorly (PL SCR from 1.2 to 2). CONCLUSION: In anatomical ACL reconstruction, the tunnel entrances are dictated by anatomy; however, there can be variations in tunnel exit positions. Consideration should be given when positioning tunnel exits on the effect on stress in the femur. Moving the PL tunnel exit laterally or posteriorly increases in the stress at the PL tunnel exit.

The effect of platelet-rich plasma on the regenerative therapy of muscle derived stem cells for articular cartilage repair.

OBJECTIVE: Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. METHODS: MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. RESULTS: The addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. CONCLUSIONS: Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.

First Report of Coleus blumei viroid 5 from Coleus blumei in India and Indonesia.

Coleus blumei, which was found originally in Indonesia, is an ornamental plant grown worldwide. It can be infected by several viroids of the genus Coleviroid, family Pospiviroidae. Six main viroids that infect coleus have been reported: Coleus blumei viroid 1 through 6 (CbVd-1 ~ CbVd-6). Although CbVd-1 was first reported in a commercial coleus in Brazil in 1989 (1), and then in Germany, Japan, Canada, Korea, China, and India, CbVd-5 was reported only in China in 2009 (2). Symptoms caused by CbVd-5 varied depending on different cultivars, and in case of an unknown cultivar of "Red with dark green edge," are very clear albino symptoms. From 2010 to 2011, 60 and 3 leaf samples of coleus were collected from Hyderabad, India, and Java, Indonesia, respectively, and subjected to low molecular weight RNA extraction according to Li et al. (3). The results of dot-blot hybridization using CbVd-5 cRNA probes and RT-PCR using CbVd-5 specific primers (CbVd-5-PF: 5'-TGACTAGAACAGTAGTAAAG-3' / CbVd-5-PR: 5'-AATTGAGGTCAAACCTCTTT-3') demonstrated that 28 out of the 60 samples from India and all three samples from Indonesia were positive for CbVd-5. The resulting RT-PCR fragments from one sample selected randomly from each country were cloned into the pMD18-T vector (Takara) and transformed into E. coli DH5α competent cells. Five positive clones of each sample were sequenced. The result of sequence analysis revealed that the similarities of CbVd-5 between the sequences we obtained and the reference sequence (GenBank Accession No. NC003683) were 97.8 to 100%. Bioassay using nine viroid-free coleus plants from three cultivars (three from each cultivar), inoculated with CbVd-5 infectious clones by stem slashing, demonstrated that CbVd-5 could induce albino symptom on the leaves of the unknown cultivar "Red with dark green edge" 2 months after inoculation. To our knowledge, this is the first report of CbVd-5 from India and Indonesia, and the second report of CbVd-5 in the world. Considering the effect of CbVd-5 on the appearance of coleus and its recombination ability, a certification program may be needed to control the spread of this viroid. References: (1) M. E. N. Fonseca et al. Fitopatol. Bras. 14:94, 1989. (2) W. Y. Hou et al. Arch. Virol. 154:315, 2009. (3) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995.

AAV-based shRNA silencing of NF-κB ameliorates muscle pathologies in mdx mice.

Chronic inflammation, promoted by an upregulated NF-kappa B (NF-κB) pathway, has a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Blocking the NF-κB pathway has been shown to be a viable approach to diminish chronic inflammation and necrosis in the dystrophin-defective mdx mouse, a murine DMD model. In this study, we used the recombinant adeno-associated virus serotype 9 (AAV9) carrying an short hairpin RNA (shRNA) specifically targeting the messenger RNA of NF-κB/p65 (p65-shRNA), the major subunit of NF-κB associated with chronic inflammation in mdx mice. We examined whether i.m. AAV9-mediated delivery of p65-shRNA could decrease NF-κB activation, allowing for amelioration of muscle pathologies in 1- and 4-month-old mdx mice. At 1 month after treatment, NF-κB/p65 levels were significantly decreased by AAV gene transfer of p65-shRNA in the two ages of treatment groups, with necrosis significantly decreased compared with controls. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67 to 34%, but the level of CN was not significantly decreased in treated 4-month-old mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Taken together, these findings demonstrate that the AAV-mediated delivery of p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by selectively reducing NF-κB/p65 activity.

Salvianolic acid A exerts antiamnesic effect on diazepam-induced anterograde amnesia in mice.

Benzodiazepine was known to produce amnesia. Salvianolic acid A extracted from Salvia miltiorrhiza was an effective antioxidant. The objective of the study was to evaluate the effect of salvianolic acid A on diazepam-induced amnesia in mice. C57BL/6 mice were treated with salvianolic acid A at doses of 10, 20 and 40 mg/kg following administration with diazepam at a dose of 3 mg/kg. Morris water maze was performed to evaluate the effect of salvianolic acid A on amnesia. The antioxidative parameters in hippocampus were measured. The results showed that salvianolic acid A decreased the mean escape latency and increased the percentage of time spent in target quadrant. Salvianolic acid A reduced the content of malondialdehyde and increased the activities of superoxide dismutase, catalase and glutathione peroxidase in hippocampus. The findings demonstrated that salvianolic acid A had antiamnesic effects on diazepam-induced anterograde amnesia in mice, by augmenting the antioxidative capacity of hippocampus.

Resting and post-exercise serum biomarkers of cardiac and skeletal muscle damage in adolescent runners.

This study examined the response of serum biomarkers of cardiac and skeletal muscle damage at rest and after a routine workout of 21 km run in 12 male adolescent (16.2±0.6 years) long-distance runners. Biomarkers of cardiac [troponins (cTnT, cTnI), creatine kinase MB mass (CK-Mbmass)] and skeletal muscle [creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hydroxybutyrate dehydrogenase (HBD)] damage were assayed at rest, 2, 4 and 24 h post-exercise. At rest, cTnT and cTnI were not detectable; however, CK, CK-MBmass, AST, ALT and HBD were above corresponding clinical cut-off values. Post-exercise significant elevations above rest were observed for all biomarkers, except ALT, 2 and 4 h following the run, and remained elevated in cTnI, CK, CK-MBmass, LDH and AST 24 h post-workout. A significant increase in data points above clinical cut-off values from rest to post-exercise was reported for cTnT, cTnI and CK at 2 and 4 h, and in cTnI and CK 24 h post-exercise. In conclusion, a 21 km run in adolescent runners increased post-exercise biomarkers of cardiac and skeletal muscle damage.

First Report of Coleus blumei viroid 2 from Commercial Coleus in China.

Coleus (Coleus blumei) is an ornamental plant that is susceptible to infection by several viroids of the genus Coleviroid, which is a member of the family Pospiviroidae. Coleus blumei viroid (CbVd) -1 was first reported in commercial yellow coleus fields in Brazil in 1989 (1). In addition, CbVd-2, CbVd-3, and CbVd-4 have only been detected from coleus in Germany in 1996 (4). CbVd-5 and CbVd-6 were recently identified in China (2). In March 2010, leaves were collected from 50 symptomless coleus plants from a commercial nursery in Hainan Province, China. Total RNA was extracted from the leaves (3). Reverse transcription (RT)-PCR using CbVd-2 specific primers (forward: 5'-AGCTTACCTGGGTTCCCT-3' and reverse: 5'-CTCTCCTCTATTTACTCTCTTCTC-3') corresponding to positions 76 to 93 and 52 to 75 on the CbVd-2 reference sequence, respectively (GenBank Accession No. NC003682). Amplification of a 301-bp product was obtained from one sample. This PCR product was then cloned into pMD18-T (Takara, Dalian, China). Twelve positive clones were sequenced and the results were subjected to BLAST analysis. Sequence analysis showed that two sequences (GenBank Accessions Nos. HQ727542 and HQ727544) shared 99% identity with the reference sequence of CbVd-2 (NC003682), and four sequences (HQ727541, HQ727543, HQ727545 and HQ727547) had 99.34% identity with the reference sequence of CbVd-2 (NC003682). The proposed secondary structures of these variants have approximately 75% paired nucleotides. Results suggested the presence of CbVd-2, which is a member of the Coleviroid genus, Pospoviroidae family. To our knowledge, this is the first report of CbVd-2 from commercial coleus in China. References: (1) M. E. N. Fonseca et al. Fitopatol. Bras. 14:94, 1989. (2) W. Y. Hou et al. Arch. Virol. 154:993, 2009. (3) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995. (4) R. L. Spieker et al. J. Gen. Virol. 77:2839, 1996.

Long-term but not short-term aspirin treatment attenuates diabetes-associated learning and memory decline in mice.

Increasing studies have shown that the patients with diabetes mellitus have an increased risk of cognitive impairment, dementia, and neurodegeneration. The present study was designed to evaluate the effect of aspirin on diabetes-associated learning and memory decline in mice. Diabetes was induced by a single intraperitoneal injection of streptozocin (150 mg/kg body weight) in C57BL/6 mice. The mice were administered with aspirin at a dose of 30 mg/kg by intragastric administration once a day for 1, 4 or 8 weeks respectively. 8 weeks after aspirin or vehicle treatment, the effect of aspirin on diabetes-associated learning and memory decline in mice was investigated by evaluating the mean escape latency and the percentage of time spent in target quadrant. The TNF-α, IL-1ß contents, and acetylcholinesterase activity in hippocampus were assayed as well. The results showed that administration with aspirin for 4 weeks or 8 weeks significantly reduced the mean escape latency, the acetylcholinesterase activity, the TNF-α, IL-1ß levels and increased the percentage of time spent in target quadrant. However, treatment with aspirin for 1 week did not ameliorate diabetes-associated learning and memory decline. The present study demonstrated that long-term aspirin treatment attenuates diabetes-associated learning and memory decline in mice, and that the effect of aspirin was related to its anti-inflammatory potency.

Cardioprotection with forsythoside B in rat myocardial ischemia-reperfusion injury: relation to inflammation response.

The present study was undertaken to examine the effect of forsythoside B (FB) on rat myocardial ischemia-reperfusion (I/R) model and elucidate the potential mechanism. Left ventricular systolic pressure (LVSP) and +/-dp/dt(max) were detected. Blood samples were collected to determine serum levels of troponin T (Tn-T), TNF-alpha and IL-6. Hearts were harvested to assess histopathological change and infarct size, determine content of MDA, myeloperoxidase (MPO), SOD and GPx activities, analyze expression of high-mobility group box 1 (HMGB1), phosphor-I kappaB-alpha and phosphor-nuclear factor kappaB (NF-kappaB) in ischemic myocardial tissue by Western blot. Compared with control group, rats treatment with FB showed a significant recovery in myocardial function with improvement of LVSP and +/-dp/dt(max). The myocardial infarct volume, serum levels of Tn-T, TNF-alpha and IL-6, content of MDA and MPO activity in myocardial tissue were all reduced, protein expression of HMGB1, phosphor-I kappaB-alpha and phosphor-NF-kappaB were down-regulated, while attenuated the decrease of SOD and GPx activities. Besides, the infiltration of polymorph nuclear leukocytes (PMNs) and histopathological damages in myocardium were decreased in FB treated groups. These findings suggested that FB rescued cardiac function from I/R injury by limiting inflammation response and its antioxidant properties.

Arthroscopic evaluation of the ACL double bundle structure.

In order to describe the arthroscopic presence of the double bundle structure and to evaluate the value of different portals in knee arthroscopy, we assessed the AM and PL bundle anatomy. We prospectively examined the knees of 60 patients undergoing arthroscopic surgery for pathology unrelated to the ACL. Arthroscopy was performed in a two portal technique using an anterolateral (ALP) and an anteromedial (AMP) portal. With the arthroscope in the ALP, we could distinguish an AM and PL bundle in 28%. Switching the arthroscope to the AMP, differentiation of the bundles was possible in 67%. In all remaining cases visualization of the PL bundle was possible after retraction of the AM bundle. Use of AMP increased visualization of the PL bundle. It seems reasonable to perform arthroscopy for ACL reconstruction with the arthroscope in the AMP and to establish an additional medial working portal to increase the visualization of the femoral ACL insertion sites for optimal femoral tunnel placement.

Aspirin attenuates cerebral ischemic injury in diabetic rats.

Diabetes is an independent risk factor for ischemic stroke. Large randomized trials have shown that aspirin reduces stroke risks in patients with diabetes. However, there was no study that reported whether aspirin could attenuate the cerebral ischemic injury when administered prior to cerebral ischemia in diabetes. The objective of the present study was to evaluate the effects of aspirin on focal cerebral ischemia in diabetic rats. Diabetic rats received an oral administration of aspirin for 7 d prior to be subjected to a permanent middle cerebral artery occlusion (MCAO). The infarct volume of the brain was assessed in brain slices stained with 2% solution of triphenyltetrazolium chloride. Behavioral tests were used to evaluate the damage to the central nervous system. Platelet aggregation induced by adenosine diphosphate was measured. The results showed that aspirin at a dose of 30 mg/kg but not 10 mg/kg significantly reduced infarct volume and decreased neurological deficit scores compared with vehicle treatment. Aspirin (30 mg/kg) treatment also reduced platelet aggregation. Administration of aspirin did not alter the levels of blood glucose and insulin in diabetic rats. The findings suggest that pretreatment with aspirin may be effective to attenuate cerebral ischemic injury in diabetic patients.

Construction and analysis of compact muscle-specific promoters for AAV vectors.

Adeno-associated viral (AAV) vectors have been broadly used for gene transfer in vivo for various applications. However, AAV precludes the use of most of the original large-sized tissue-specific promoters for expression of transgenes. Efforts are made to develop highly compact, active and yet tissue-specific promoters for use in AAV vectors. In this study, we further abbreviated the muscle creatine kinase (MCK) promoter by ligating a double or triple tandem of MCK enhancer (206-bp) to its 87-bp basal promoter, generating the dMCK (509-bp) and tMCK (720-bp) promoters. The dMCK promoter is shorter but stronger than some previously developed MCK-based promoters such as the enh358MCK (584-bp) and CK6 (589-bp) in vitro in C2C12 myotubes and in vivo in skeletal muscles. The tMCK promoter is the strongest that we tested here, more active than the promiscuous cytomegalovirus (CMV) promoter. Furthermore, both the dMCK and tMCK promoters are essentially inactive in nonmuscle cell lines as well as in the mouse liver (>200-fold weaker than the CMV promoter). The dMCK promoter was further tested in a few lines of transgenic mice. Expression of LacZ or minidystrophin gene was detected in skeletal muscles throughout the body, but was weak in the diaphragm, and undetectable in the heart and other tissues. Similar to other miniature MCK promoters, the dMCK promoter also shows preference for fast-twitch myofibers. As a result, we further examined a short, synthetic muscle promoter C5-12 (312-bp). It is active in both skeletal and cardiac muscles but lacks apparent preference on myofiber types. Combination of a MCK enhancer to promoter C5-12 has increased its strength in muscle by two- to threefold. The above-mentioned compact muscle-specific promoters are well suited for AAV vectors in muscle-directed gene therapy studies.