Carnosic Acid Inhibits Herpes Simplex Virus Replication by Suppressing Cellular ATP Synthesis.

Acquiring resistance against antiviral drugs is a significant problem in antimicrobial therapy. In order to identify novel antiviral compounds, the antiviral activity of eight plants indigenous to the southern region of Hungary against herpes simplex virus-2 (HSV-2) was investigated. The plant extracts and the plant compound carnosic acid were tested for their effectiveness on both the extracellular and intracellular forms of HSV-2 on Vero and HeLa cells. HSV-2 replication was measured by a direct quantitative PCR (qPCR). Among the tested plant extracts, Salvia rosmarinus (S. rosmarinus) exhibited a 90.46% reduction in HSV-2 replication at the 0.47 µg/mL concentration. Carnosic acid, a major antimicrobial compound found in rosemary, also demonstrated a significant dose-dependent inhibition of both extracellular and intracellular forms of HSV-2. The 90% inhibitory concentration (IC90) of carnosic acid was between 25 and 6.25 µg/mL. Proteomics and high-resolution respirometry showed that carnosic acid suppressed key ATP synthesis pathways such as glycolysis, citrate cycle, and oxidative phosphorylation. Inhibition of oxidative phosphorylation also suppressed HSV-2 replication up to 39.94-fold. These results indicate that the antiviral action of carnosic acid includes the inhibition of ATP generation by suppressing key energy production pathways. Carnosic acid holds promise as a potential novel antiviral agent against HSV-2.

External modulation of Rotimer exudate secretion in monogonant rotifers.

The Rotimer, a rotifer-specific biopolymer, is an exogenic bioactive exudate secreted by different monogonant species (e.g. Euchlanis dilatata or Lecane bulla). The production of this viscoelastic biomolecule is induced by different micro-particles, thereby forming a special Rotimer-Inductor Conglomerate (RIC) in a web format. In this case, the water insoluble Carmine crystals, filtered to size (max. diameter was 50 µm), functioned as an inductor. The RIC production is an adequate empirical indicator to follow up this filamentous biopolymer secretion experientially; moreover, this procedure is very sensitive to the environmental factors (temperature, pH, metals and possible natural pollutant agents). The above mentioned species show completely different reactions to these factors, except to the presence of calcium and to the modulating effects of different drugs. One of the novelties of this work is that the Rotimer secretion and consequently, the RIC-formation is a mutually obligatory and evolutionary calcium-dependent process in the concerned monogonants. This in vivo procedure needs calcium, both for the physiology of animals and for fiber formation, particularly in the latter case. The conglomerate covered area (%) and the detection of the longest filament (mm) of the given RIC were the generally and simultaneously applied methods in the current modulating experiments. Exploring the regulatory (e.g. calcium-dependency) and stimulating (e.g. Lucidril effect) possibilities of biopolymer secretion are the basis for optimizing the RIC-production capacities of these micro-metazoans.

Molecular characterization of metronidazole resistant Bacteroides strains from Kuwait.

Eleven metronidazole resistant Bacteroides and one newly classified Phocaeicola dorei strain from Kuwait were investigated for their resistance mechanisms and the emergence of their resistant plasmids. All but one strain harbored nimE genes on differently sized plasmids. Of the 11 nimE genes, 9 were preceded by full copies of the prototype ISBf6 insertion sequence element, one carried a truncated ISBf6 and one was activated by an additional copy of IS612B. Nucleotide sequencing results showed that the nimE ISBf6 distances were constant and all five different plasmids shared a common region, suggesting that (i) the nimE-ISBf6 configuration was inserted into an undisclosed common genetic element, (ii) over time, this common element was mutated by insertions and deletions, spreading the resultant plasmids. Of the 10 B. fragilis strains in this collection, 6 were also cfiA-positive, one with full imipenem resistance, indicating a tendency for multidrug resistance (MDR) among such isolates. The significant number of metronidazole resistant Bacteroides spp. and P. dorei strains with the MDR phenotype warns of difficulties in treatment and suggests promoting adherence to antibiotic stewardship recommendations in Kuwait.

GENETIC POLYMORPHISMS OF HUMAN ß-DEFENSINS IN PATIENTS WITH MULTIPLE SCLEROSIS.

AIMS: Recent studies have started to elucidate the contribution of microbiome to the pathogenesis of multiple sclerosis (MS). It is also supposed, that neuropathological alterations might be associated with abnormal expression and regulatory function of antimicrobial peptides (AMPs), including defensins. It is in our interest to investigate the relevance of the single nucleotide polymorphisms (SNPs) of the DEFB1 gene and the copy number polymorphism of the DEFB4 genes in MS. METHODS: DEFBI polymorphisms: c.-20G > A (rsl 1362), DEFB1 c.-44C > G (rsI 800972), DEFB1 c.-52G>A (rsl 799946), and the DEFB4 gene copy number were investigated in 250 MS patients The control patients comprised 232 age- and gender-matched healthy blood donors. The occurrence of the human ß-defensin 2 peptide (hBD2) in the plasma of controls and patients-was determined by ELISA. RESULTS: The DEFB1 c.-44C>G polymorphism the GG protective genotype was much less frequent among patients than among the controls. A higher frequency of a lower (<4) copy number of the DEFB4 gene was observed in the patients with MS as compared with the controls (43% vs. 28%, respectively). The median levels of the circulating hBD2 in the patients were 150.6 +/- 12.71 pg/ml vs. 262.1 +/- 23.82 pg/mI in the control group (p<0.0001). Our results suggest that ß-defensins play role in the development of MS.

A novel, multiplex, real-time PCR-based approach for the detection of the commonly occurring pathogenic fungi and bacteria.

BACKGROUND: Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified.The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. RESULTS: A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons.With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube. CONCLUSIONS: This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice.

Isocitrate lyase encoding plasmids in BCG cause increased survival in ApoB100-only LDLR-/- mice.

We studied the role of isocitrate lyase in the interaction between Mycobacterium bovis BCG and mice. ApoB100-only LDLR-/- (B6;129S-ApoBtm2SgyLdlrtm1Her/J) mice were inoculated with M. bovis BCG harbouring plasmids carrying the gene for isocitrate lyase. The presence of ~29 times more copies of this gene resulted in a higher bacterial yield in the spleens and lungs of the infected mice. The spleen was 3-4 times heavier, and in the spleen the bacteria survived over 10 days longer than did the bacteria with the control plasmid. Propionate was less toxic for bacteria carrying icl plasmids in vitro. This recombinant BCG can be a possible vaccine candidate.

Phenotypic and Molecular Characterization of Carbapenem-Heteroresistant Bacteroides fragilis Strains.

Carbapenem-resistant Bacteroides fragilis strains usually emerge by an insertion sequence (IS) jump into the upstream region of the cfiA carbapenemase gene. However, intermediate or fully resistant cfiA-positive strains also exist. These do not have such IS element activations, but usually have heterogeneous resistance (HR) phenotypes, as detected by a disc diffusion or gradient tests. Heteroresistance is a serious antibiotic resistance problem, whose molecular mechanisms are not fully understood. We aim to characterize HR and investigate diagnostic issues in the set of cfiA-positive B. fragilis strains using phenotypic and molecular methods. Of the phenotypic methods used, the population analysis profile (PAP) and area under curve (AUC) measurements were the best prognostic markers for HR. PAP AUC, imipenem agar dilution and imipenemase production corresponded well with each other. We also identified a saturation curve parameter (quasi-PAP curves), which correlated well with these phenotypic traits, implying that HR is a stochastic process. The genes, on a previously defined 'cfiA element', act in a complex manner to produce the HR phenotype, including a lysine-acetylating toxin and a lysine-rich peptide. Furthermore, imipenem HR is triggered by imipenem. The two parameters that most correlate with the others are imipenemase production and 'GNAT' expression, which prompted us to suspect that carbapenem heteroresistance of the B. fragilis strains is stochastically regulated and is mediated by the altered imipenemase production.

Single Nucleotide Polymorphisms of Indoleamine 2,3-Dioxygenase 1 Influenced the Age Onset of Parkinson's Disease.

BACKGROUND: Earlier studies reported alterations of the kynurenine (KYN) pathway of tryptophan (TRP) metabolism in Parkinson's disease (PD). The first rate-limiting enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan dioxygenase were observed upregulated, resulting elevated KYN/TRP ratios in the serum and cerebrospinal fluid samples of patients with PD. More and more single nucleotide polymorphisms (SNPs) have been identified in a population of PD. However, little is known about the impact of genetic variations of the IDO on the pathogenesis of PD. METHODS: SNP analysis of IDO1 was performed by allelic discrimination assay with fluorescently labelled TaqMan probes and a subgroup analysis was conducted according to the age of PD onset. The frame shifts variant rs34155785, intronic variant rs7820268, and promotor region variant rs9657182 SNPs of 105 PD patients without comorbidity were analyzed and compared to 129 healthy controls. RESULTS: No significant correlation was found in three SNPs between PD patients and healthy controls. However, the subgroup analysis revealed that A alleles of rs7820268 SNP or rs9657182 SNP carriers contribute to later onset of PD than non-carriers. CONCLUSIONS: The study suggested that SNPs of IDO1 influenced the age onset of PD and genotyping of SNPs in certain alleles potentially serves as a risk biomarker of PD.

Decoding Covid-19 with the SARS-CoV-2 Genome.

PURPOSE OF REVIEW: SARS-CoV-2, the recently emerged coronavirus (CoV) that is responsible for the current global pandemic Covid-19, first appeared in late 2019 in Wuhan, China. Here, we summarise details of the SARS-CoV-2 genome to assist understanding of the emergence, evolution and diagnosis of this deadly new virus. RECENT FINDINGS: Based on high similarities in the genome sequences, the virus is thought to have arisen from SARS-like CoVs in bats but the lack of an intermediate species containing a CoV with even greater similarity has so far eluded discovery. The critical determinant of the SARS-CoV-2 genome is the spike (S) gene encoding the viral structural protein that interacts with the host cell entry receptor ACE2. The S protein is sufficiently adapted to bind human ACE2 much more readily than SARS-CoV, the most closely related human CoV. SUMMARY: Although the SARS-CoV-2 genome is undergoing subtle evolution in humans through mutation that may enhance transmission, there is limited evidence for attenuation that might weaken the virus. It is also still unclear as to the events that led to the virus' emergence from bats. Importantly, current diagnosis requires specific recognition and amplification of the SARS-CoV-2 RNA genome by qPCR, despite these ongoing viral genome changes. Alternative diagnostic procedures relying on immunoassay are becoming more prevalent.

The Relationship between Antibiotic Susceptibility and pH in the Case of Uropathogenic Bacteria.

Urinary tract infections (UTIs) are common bacterial infections caused mainly by enteric bacteria. Numerous virulence factors assist bacteria in the colonization of the bladder. Bacterial efflux pumps also contribute to bacterial communication and to biofilm formation. In this study, the phenotypic and genetic antibiotic resistance of clinical UTI pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis were determined by disk diffusion method and polymerase chain reaction (PCR). Following this, different classes of antibiotics were evaluated for their antibacterial activity at pH 5, 6, 7 and 8 by a microdilution method. Gentamicin (GEN) was the most potent antibacterial agent against E. coli strains. The effect of GEN on the relative expression of marR and sdiA genes was evaluated by quantitative PCR. The slightly acidic pH (pH 6) and GEN treatment induced the upregulation of marR antibiotic resistance and sdiA QS activator genes in both E. coli strains. Consequently, bacteria had become more susceptible to GEN. It can be concluded that antibiotic activity is pH dependent and so the artificial manipulation of urinary pH can contribute to a more effective therapy of multidrug resistant bacterial infections.

Distinct changes in chronic pain sensitivity and oxytocin receptor expression in a new rat model (Wisket) of schizophrenia.

Clinical studies have shown that schizophrenia is accompanied by hypoalgesia. Accordingly, we have previously reported that a chronic schizophrenia-related rat substrain (Wisket) showed decreased acute heat pain sensitivity. The aim of the present study was to determine the mechanical pain sensitivity and the effects of opioid ligands in a chronic osteoarthritic pain model generated using Wisket rats. Our previous molecular biological studies indicated that the impairment in opioid and cannabinoid receptor functions observed in these animals did not explain their altered pain sensitivity. Therefore, we aimed to investigate another endogenous antinociceptive system, i.e., the oxytocinergic system (which is also implicated in schizophrenia) via the determination the brain-region specific oxytocin receptor mRNA expression in Wisket rats. Osteoarthritis was induced in male adult control Wistar rats without any interventions and in Wisket rats after juvenile social isolation and ketamine treatment. The degree of allodynia and the effects of systemic morphine or intrathecal endomorphin-1 administration were determined. Furthermore, the expression of the oxytocin receptor mRNA was assessed in different brain structures (prefrontal cortex, striatum, diencephalon, brainstem, and olfactory bulb). A lower degree of allodynia was observed in the Wisket group compared with control animals 1 and 2 weeks after the induction of osteoarthritis, which was accompanied by a comparable degree of edema. Systemically or intrathecally applied opioids caused similar time-response curves in both groups, with apparently shorter effects in Wisket animals. The expression of the oxytocin receptor mRNA was lower in most of the brain regions (with the exception of the diencephalon) investigated in Wisket rats vs. the control animals. In summary, both acute and chronic hypoalgesia (as nonspecific symptoms in patients with schizophrenia) can be simulated in Wisket animals as endophenotypes despite the impairment of the endogenous antinociceptive systems evaluated. Thus, this model might be an appropriate tool for further investigation of the molecular basis of altered pain perception in schizophrenia.

Correlation between detergent activity and anti-herpes simplex virus-2 activity of commercially available vaginal gels.

OBJECTIVE: Herpes simplex virus-2 (HSV-2) infections are almost exclusively sexually transmitted. The presence of vaginal gels during sexual activity may have a significant positive or negative impact on viral transmission. Therefore we investigated three off-the-shelf vaginal lubricants and one pH restoring gel to evaluate their impact on HSV-2 replication. RESULTS: HeLa cells were infected with untreated virions and virions incubated with the particular gels. The accumulation of viral genomes was monitored by quantitative PCR (qPCR) method at 24 h post infection. Two of the tested gels had no significant effect on HSV-2 replication at the maximum applied concentration, while two had a strong inhibitory effect (~ 98% reduction of replication). The replication inhibitory effect was observed at various multiplicity of infection (MOI 0.4-6.4) and the two inhibitory gels were also capable of inhibiting the HSV-2 induced cytopathic effect on HeLa cells. The surface tension decreasing activity-an indication of detergent activity-was strongly correlated with the anti-HSV-2 activity of the gels (R2: 0.88). Our results indicate that off-the-shelf vaginal gels have a markedly different anti-HSV-2 activity that may influence HSV-2 transmission.

Characteristic imprint of single nucleotide polymorphisms in multiple sclerosis.

Although the main pathomechanism of multiple sclerosis (MS) is not known, an autoimmune response against the myelin basic proteins (MBPs) is presumed to be involved in its evolution and propagation. In this study, we examined whether the nucleotide sequences of the 3' untranslated regions (UTRs) of the DNAs encoding the MBP are characteristic of MS. These genetic regions are presumed to be responsible for the transport and localization of the mRNAs encoding the MBP in the glia cells, thereby influencing the building up of the myelin sheaths of the glia cells. The DNA region involving nucleotides 710-1540 of the UTRs of the MBPs was sequenced and analyzed in 52 relapsing-remitting MS patients, in 52 neuroimaging alteration-free controls, and in 45 healthy volunteers. Although the examined UTRs exhibited a wide range of sequence variations in both the MS and the control subjects, we found a typical distribution of single nucleotide polymorphisms (SNPs) along the examined DNA sequence in the MS patients, which was different from that in the controls. We could distinguish two genetic regions: region A--nucleotide positions 851-896 and B--nucleotide positions 897-1540, in the UTRs. Subjects with SNPs in region A but without SNPs in region B occurred significantly more frequently in the MS group than in the control group (30.8% versus 3.85%, p < 0.0002848, OR 11.11, 95% CI--2.4-51.4, p < 0.0004). The distribution pattern of the SNPs in the UTRs seems to be highly characteristic of relapsing-remitting MS. These findings call attention to the possible roles of the UTRs of the MBPs in the development of MS.

Cloning of the Rhizomucor miehei 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in Mucor circinelloides.

In this study, the gene hmgR encoding the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) was cloned and characterized in the zygomycete fungus Rhizomucor miehei. The hmgR gene comprises a total of 3,585 bp including the coding sequence of a 1,058 amino acids length putative protein and five introns (137, 83, 59, 60 and 69 bp in length) dispersed in the whole coding region. Southern hybridization analysis revealed that the gene is present only in one copy in the R. miehei genome. The isolated Rhizomucor gene was expressed in the related fungus, Mucor circinelloides. Transformants harbouring the Rhizomucor hmgR gene in an autoreplicative plasmid proved to be more tolerant to statins (e.g. lovastatin, simvastatin, and fluvastatin), the competitive inhibitors of the HMG-CoA reductase, than the original M. circinelloides strain. At the same time, heterologous expression of the Rhizomucor hmgR did not affect the carotenoid production of M. circinelloides.

Evaluation of the genetic variants of kinesin motor protein in ischemic stroke.

BACKGROUND: The kinesin light-chain 1 genetic variants G56836C, A185C, and C406T were earlier found to amplify the development of leukoaraiosis in hypertensive smokers. These 3 variants were presumed to affect the function of the mitochondria, thereby giving rise to sensitivity to a chronic ischemic state. We have now extended our investigations to examine how the above genetic variants affect the occurrence of ischemic stroke. METHODS: Genetic and clinical data on 650 ischemic stroke and 340 neuroimaging alteration-free subjects were analyzed. Univariate and logistic regression approaches were used. RESULTS: None of the above genetic variants proved to be risk factors of ischemic stroke, either alone or in combination with other clinical factors. CONCLUSION: The examined 3 genetic variants seem to influence the responses of the glial cells to a slight chronic hypoxia state, rather than the mechanisms resulting in cerebral infarcts themselves.

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures.

OBJECTIVE: We aim to directly detect Mycoplasma DNA in a U937 suspension cell culture without using DNA purification. In order to make Mycoplasma contamination monitoring easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit. We compared the sensitivity of direct qPCR against qPCR with a purified DNA template. RESULTS: Our findings indicate that qPCR worked optimally with a 6 µl sample volume and a 52 °C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 to 20 s without any major decrease in reaction sensitivity. The total cycle time of optimized direct qPCR was 65 min. The optimized qPCR protocol was used to detect Mycoplasma DNA before and after DNA purification. Our findings indicate that direct qPCR had a higher sensitivity than regular qPCR. Ct levels produced by direct qPCR with 6 µl templates were almost identical to Ct levels produced by regular qPCR with DNA purified from a 60 µl cell culture sample (23.42 vs 23.49 average Ct levels, respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from U937 cell cultures.

Indoleamine 2,3-Dioxygenase Activity in Chlamydia muridarum and Chlamydia pneumoniae Infected Mouse Lung Tissues.

Chlamydia trachomatis infections are the most prevalent sexually transmitted infections with potentially debilitating sequelae, such as infertility. Mouse models are generally used for vaccine development, to study the immune response and histopathology associated with Chlamydia infection. An important question regarding murine models is the in vivo identification of murine host genes responsible for the elimination of the murine and human Chlamydia strains. RNA sequencing of the Chlamydia muridarum infected BALB/c lung transcriptome revealed that several genes with direct antichlamydial functions were induced at the tissue level, including the already described and novel members of the murine interferon-inducible GTPase family, the CXCL chemokines CXCL9, CXCL11, immunoresponsive gene 1, nitric oxide synthase-2 (iNOS), and lipocalin-2. Indoleamine 2,3-dioxygenase 1-2 (IDO1-2) previously described potent antichlamydial host enzymes were also highly expressed in the infected murine lungs. This finding was novel, since IDO was considered as a unique human antichlamydial defense gene. Besides a lower level of epithelial cell positivity, immunohistochemistry showed that IDO1-2 proteins were expressed prominently in macrophages. Detection of the tryptophan degradation product kynurenine and the impact of IDO inhibition on Chlamydia muridarum growth proved that the IDO1-2 proteins were functionally active. IDO1-2 activity also increased in Chlamydia muridarum infected C57BL/6 lung tissues, indicating that this phenomenon is not mouse strain specific. Our study shows that the murine antichlamydial response includes a variety of highly up-regulated defense genes in vivo. Among these genes the antichlamydial effectors IDO1-2 were identified. The potential impact of murine IDO1-2 expression on Chlamydia propagation needs further investigation.

A cytoskeleton motor protein genetic variant may exert a protective effect on the occurrence of multiple sclerosis: the janus face of the kinesin light-chain 1 56836CC genetic variant.

Although the main pathomechanism of multiple sclerosis (MS) is not known, an autoimmune response is presumed to involve its evolution and propagation. In this study, we examined how the kinesin light-chain 1 (KLC1) G56836C (rs8702) single nucleotide polymorphism (SNP) in intron 13 affects the occurrence of MS. This genetic variant was found to be associated with cognitive disturbances and neurodegeneration, and it was presumed to affect the kinesin function. Kinesin serves as a main cytoskeleton motor protein by carrying mitochondria and the molecular apparatus of myelin basic protein synthesis. The present association analysis of this genetic variant was performed in 102 relapsing-remitting MS patients and in 207 neuroimaging alteration-free controls. The KLC1 56836CC variant proved to exert a significant protective effect on the occurrence of MS (2.0% vs. 9.7%, P < 0.02; crude OR: 0.19, 95% CI: 0.04-0.82, P < 0.05; adjusted OR: 0.21, 95% CI: 0.018-0.88, P < 0.05). Our results draw attention to possible roles of the cytoskeleton in MS.

A genetic variant in cytoskeleton motors amplifies susceptibility to leukoaraiosis in hypertensive smokers: gene-environmental interactions behind vascular white matter demyelinization.

One of the most frequent causes of an age-associated cognitive decline is the vascular white matter demyelinization of the brain referred to as leukoaraiosis (LA). The wide range of severity of the cognitive decline caused by LA can have numerous deleterious effects on the quality of life, leading overall to far-reaching public health problems. Besides clinical risk factors such as hypertension and advanced age, genetic susceptibility factors are presumed to be of great importance in the development of LA. The protein kinesin, which is the main motor protein in the trafficking system of the mitochondria, can undergo functional damage under the circumstances of chronic hypoxia. This may give rise to a slowly developing metabolic crisis in the glia cells, a phenomenon hypothesized to account for the evolution of LA. Setting out from this assumption, we examined how the kinesin light-chain 1 (KNS2) G56836C single nucleotide polymorphism in intron 13 affects the susceptibility to LA. This genetic variant was found to be associated with cognitive disturbances and neurodegeneration, and it was presumed to affect the function of kinesin. The association analysis of the above genetic variant was performed in 229 patients with LA and 264 neuroimaging alteration-free controls. The KNS2 56836CC variant increased the risk of LA 7.76-fold in hypertensive smokers as compared with those not carrying this variant. This finding may be useful in everyday clinical practice by indicating the need for stricter preventive measures in CC carriers.